<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with Wdesk from Workiva-->
<!--Copyright 2022 Workiva-->
<!--r:bb85fb30-cf0e-4708-97aa-059f6df09562,g:ab8acc49-3bf8-4304-a041-c53a61b4a79c-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://www.csx.com/role/CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnaudited" xlink:type="simple" xlink:href="csx-20220331.xsd#CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnaudited"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnaudited" xlink:type="extended" id="ic168533c6f29404ca0b1536485fe809f_CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnaudited">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_80ddec5b-9fd2-446b-801b-646a6bb64758" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_80ddec5b-9fd2-446b-801b-646a6bb64758" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_2f225e45-85b1-451d-ab36-de385fa22daa" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_2f225e45-85b1-451d-ab36-de385fa22daa" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_fd475875-23e6-4265-bd14-5325467b21c6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_fd475875-23e6-4265-bd14-5325467b21c6" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_2c652b87-4463-4104-aebb-e868898fdb7e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_2c652b87-4463-4104-aebb-e868898fdb7e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_0495ed94-7f3d-4f89-9f19-db485f6483ce" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_2c652b87-4463-4104-aebb-e868898fdb7e" xlink:to="loc_us-gaap_NetIncomeLoss_0495ed94-7f3d-4f89-9f19-db485f6483ce" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_de07ab1f-3bad-442d-a10d-eb14fb2075af" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_2c652b87-4463-4104-aebb-e868898fdb7e" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_de07ab1f-3bad-442d-a10d-eb14fb2075af" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTax_0841d912-3511-4a4f-b3e3-89372d3d69eb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ComprehensiveIncomeNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_2c652b87-4463-4104-aebb-e868898fdb7e" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTax_0841d912-3511-4a4f-b3e3-89372d3d69eb" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DividendsCommonStockCash_d58d9270-f494-4b98-baaf-124bafbad2c3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DividendsCommonStockCash"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_us-gaap_DividendsCommonStockCash_d58d9270-f494-4b98-baaf-124bafbad2c3" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodShares_0f7c657b-d78c-444b-bd94-bd582b851bf3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockRepurchasedAndRetiredDuringPeriodShares"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodShares_0f7c657b-d78c-444b-bd94-bd582b851bf3" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodValue_b5844f4d-96c1-458a-ad8b-247bfdb352c2" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockRepurchasedAndRetiredDuringPeriodValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodValue_b5844f4d-96c1-458a-ad8b-247bfdb352c2" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodAndOther_901a7809-77c4-4cc7-bfcd-90432a6edb15" xlink:href="csx-20220331.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodAndOther"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodAndOther_901a7809-77c4-4cc7-bfcd-90432a6edb15" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_StockIssuedDuringPeriodValueStockOptionsExercisedAndOther_a8ac9600-909b-492d-87f0-58e06fdc9771" xlink:href="csx-20220331.xsd#csx_StockIssuedDuringPeriodValueStockOptionsExercisedAndOther"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_8821b868-2931-4873-96e3-c21d0a9ee255" xlink:to="loc_csx_StockIssuedDuringPeriodValueStockOptionsExercisedAndOther_a8ac9600-909b-492d-87f0-58e06fdc9771" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_fd699042-1230-405e-9ca8-fb8c6c1a01c7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_83b038e6-c478-4595-975f-f2c80e7bbcd5" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AociTaxAttributableToParent_525b80eb-f078-4c6b-bc2e-d4b1a55a962b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AociTaxAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_80ddec5b-9fd2-446b-801b-646a6bb64758" xlink:to="loc_us-gaap_AociTaxAttributableToParent_525b80eb-f078-4c6b-bc2e-d4b1a55a962b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_c33d5fa1-2f55-4a2c-8362-b19abbb2479d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_80ddec5b-9fd2-446b-801b-646a6bb64758" xlink:to="loc_us-gaap_StatementTable_c33d5fa1-2f55-4a2c-8362-b19abbb2479d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_d4d90df4-14e0-4245-8221-999bb646a199" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_c33d5fa1-2f55-4a2c-8362-b19abbb2479d" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_d4d90df4-14e0-4245-8221-999bb646a199" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_d4d90df4-14e0-4245-8221-999bb646a199_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_d4d90df4-14e0-4245-8221-999bb646a199" xlink:to="loc_us-gaap_EquityComponentDomain_d4d90df4-14e0-4245-8221-999bb646a199_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_d4d90df4-14e0-4245-8221-999bb646a199" xlink:to="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_447c109f-3316-43a1-bee2-d5924a32c0b3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:to="loc_us-gaap_CommonStockMember_447c109f-3316-43a1-bee2-d5924a32c0b3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockIncludingAdditionalPaidInCapitalMember_5efa5149-3fa1-4894-aa62-cc73c8e1e7f9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommonStockIncludingAdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:to="loc_us-gaap_CommonStockIncludingAdditionalPaidInCapitalMember_5efa5149-3fa1-4894-aa62-cc73c8e1e7f9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_d280067b-0c75-4797-b354-bef87da23cca" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:to="loc_us-gaap_RetainedEarningsMember_d280067b-0c75-4797-b354-bef87da23cca" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_83542ad6-0d1e-4cb9-bbe0-d0362491ce87" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_83542ad6-0d1e-4cb9-bbe0-d0362491ce87" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NoncontrollingInterestMember_36888a66-cebc-4612-95f3-a5d486234b73" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_NoncontrollingInterestMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_7d777586-6dee-498c-beeb-3002674f34e5" xlink:to="loc_us-gaap_NoncontrollingInterestMember_36888a66-cebc-4612-95f3-a5d486234b73" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/NatureofOperationsandSignificantAccountingPoliciesDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#NatureofOperationsandSignificantAccountingPoliciesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/NatureofOperationsandSignificantAccountingPoliciesDetails" xlink:type="extended" id="icea5b1d545904d74bad47fc7e1e5ff7c_NatureofOperationsandSignificantAccountingPoliciesDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_RailRouteNetworkDistance_282945b6-a0cf-45d4-82fa-2fc058521bdd" xlink:href="csx-20220331.xsd#csx_RailRouteNetworkDistance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_csx_RailRouteNetworkDistance_282945b6-a0cf-45d4-82fa-2fc058521bdd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NumberOfStatesInWhichEntityOperates_a194eefc-c535-4e8e-b7fb-2cfe11cf6250" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_NumberOfStatesInWhichEntityOperates"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_NumberOfStatesInWhichEntityOperates_a194eefc-c535-4e8e-b7fb-2cfe11cf6250" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfPropertyPlantAndEquipmentForSale_6392d91c-1dc8-43f9-b2b0-ed909e997d15" xlink:href="csx-20220331.xsd#csx_NumberOfPropertyPlantAndEquipmentForSale"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_csx_NumberOfPropertyPlantAndEquipmentForSale_6392d91c-1dc8-43f9-b2b0-ed909e997d15" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfPhasesToCompletePropertyPlantAndEquipmentSaleTransaction_e41aa3a2-8fe1-4069-aaba-c592b152a999" xlink:href="csx-20220331.xsd#csx_NumberOfPhasesToCompletePropertyPlantAndEquipmentSaleTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_csx_NumberOfPhasesToCompletePropertyPlantAndEquipmentSaleTransaction_e41aa3a2-8fe1-4069-aaba-c592b152a999" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SaleOfPropertyPlantAndEquipmentConsideration_d5887fc0-9d7a-4f1d-a849-253420dd61c5" xlink:href="csx-20220331.xsd#csx_SaleOfPropertyPlantAndEquipmentConsideration"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_csx_SaleOfPropertyPlantAndEquipmentConsideration_d5887fc0-9d7a-4f1d-a849-253420dd61c5" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromSaleOfPropertyPlantAndEquipment_b7375225-f789-4d07-b207-a38041fe9ef8" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ProceedsFromSaleOfPropertyPlantAndEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_ProceedsFromSaleOfPropertyPlantAndEquipment_b7375225-f789-4d07-b207-a38041fe9ef8" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GainLossOnSaleOfPropertyPlantEquipment_ed77a1b9-5ff0-4a62-95e9-d38a9ab455f7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_GainLossOnSaleOfPropertyPlantEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_GainLossOnSaleOfPropertyPlantEquipment_ed77a1b9-5ff0-4a62-95e9-d38a9ab455f7" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityNoteStockSplitConversionRatio1_89154d87-71b2-431f-97ee-052bd6427242" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockholdersEquityNoteStockSplitConversionRatio1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_StockholdersEquityNoteStockSplitConversionRatio1_89154d87-71b2-431f-97ee-052bd6427242" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_StockholdersEquityNoteStockSplitNumberOfAdditionalShares_aa26c81c-18c3-4af3-85c3-fac61f6413b9" xlink:href="csx-20220331.xsd#csx_StockholdersEquityNoteStockSplitNumberOfAdditionalShares"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_csx_StockholdersEquityNoteStockSplitNumberOfAdditionalShares_aa26c81c-18c3-4af3-85c3-fac61f6413b9" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsAccumulatedDeficit_492efa11-ffa4-40e2-b7e3-db0f2e2ca1cc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetainedEarningsAccumulatedDeficit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_RetainedEarningsAccumulatedDeficit_492efa11-ffa4-40e2-b7e3-db0f2e2ca1cc" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalCommonStock_84a83819-a977-48db-b0d7-ece4f2069efa" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AdditionalPaidInCapitalCommonStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_AdditionalPaidInCapitalCommonStock_84a83819-a977-48db-b0d7-ece4f2069efa" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfEntityWideInformationRevenueFromExternalCustomersByProductsAndServicesTable_4f57c657-0780-49da-aa8a-ff5f6c773138" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfEntityWideInformationRevenueFromExternalCustomersByProductsAndServicesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_EntityWideInformationRevenueFromExternalCustomerLineItems_cf6a5176-5911-49c6-b6cd-ee71506dd36e" xlink:to="loc_us-gaap_ScheduleOfEntityWideInformationRevenueFromExternalCustomersByProductsAndServicesTable_4f57c657-0780-49da-aa8a-ff5f6c773138" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_d9f10434-33a8-450f-9cdb-8c52bb4dcc00" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfEntityWideInformationRevenueFromExternalCustomersByProductsAndServicesTable_4f57c657-0780-49da-aa8a-ff5f6c773138" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_d9f10434-33a8-450f-9cdb-8c52bb4dcc00" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_d9f10434-33a8-450f-9cdb-8c52bb4dcc00_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_d9f10434-33a8-450f-9cdb-8c52bb4dcc00" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_d9f10434-33a8-450f-9cdb-8c52bb4dcc00_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_e6572f1e-344d-466c-91a0-5c6daf9b4a1e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_d9f10434-33a8-450f-9cdb-8c52bb4dcc00" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_e6572f1e-344d-466c-91a0-5c6daf9b4a1e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LandEasementMember_f2df0ad7-6186-428b-900a-5179071575e6" xlink:href="csx-20220331.xsd#csx_LandEasementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_e6572f1e-344d-466c-91a0-5c6daf9b4a1e" xlink:to="loc_csx_LandEasementMember_f2df0ad7-6186-428b-900a-5179071575e6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RestatementAxis_82b9084b-b076-433a-bf47-dfe3104ceb56" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RestatementAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfEntityWideInformationRevenueFromExternalCustomersByProductsAndServicesTable_4f57c657-0780-49da-aa8a-ff5f6c773138" xlink:to="loc_srt_RestatementAxis_82b9084b-b076-433a-bf47-dfe3104ceb56" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RestatementDomain_82b9084b-b076-433a-bf47-dfe3104ceb56_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RestatementDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RestatementAxis_82b9084b-b076-433a-bf47-dfe3104ceb56" xlink:to="loc_srt_RestatementDomain_82b9084b-b076-433a-bf47-dfe3104ceb56_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RestatementDomain_7f99507d-da53-4e5e-9614-712af85d7ffd" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RestatementDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RestatementAxis_82b9084b-b076-433a-bf47-dfe3104ceb56" xlink:to="loc_srt_RestatementDomain_7f99507d-da53-4e5e-9614-712af85d7ffd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RevisionOfPriorPeriodReclassificationAdjustmentMember_2296713f-0a34-4ea2-a96d-ed965f0153ef" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RevisionOfPriorPeriodReclassificationAdjustmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RestatementDomain_7f99507d-da53-4e5e-9614-712af85d7ffd" xlink:to="loc_srt_RevisionOfPriorPeriodReclassificationAdjustmentMember_2296713f-0a34-4ea2-a96d-ed965f0153ef" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationTypeAxis_db693b3d-a43d-4efe-9a5a-0968f568c503" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReclassificationTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfEntityWideInformationRevenueFromExternalCustomersByProductsAndServicesTable_4f57c657-0780-49da-aa8a-ff5f6c773138" xlink:to="loc_us-gaap_ReclassificationTypeAxis_db693b3d-a43d-4efe-9a5a-0968f568c503" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationTypeDomain_db693b3d-a43d-4efe-9a5a-0968f568c503_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReclassificationTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ReclassificationTypeAxis_db693b3d-a43d-4efe-9a5a-0968f568c503" xlink:to="loc_us-gaap_ReclassificationTypeDomain_db693b3d-a43d-4efe-9a5a-0968f568c503_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationTypeDomain_c403e8b6-ebbe-44b1-b11a-5fa589019331" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReclassificationTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ReclassificationTypeAxis_db693b3d-a43d-4efe-9a5a-0968f568c503" xlink:to="loc_us-gaap_ReclassificationTypeDomain_c403e8b6-ebbe-44b1-b11a-5fa589019331" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationOtherMember_36b1eeba-15a8-4e92-9ce8-c444491f808a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReclassificationOtherMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationTypeDomain_c403e8b6-ebbe-44b1-b11a-5fa589019331" xlink:to="loc_us-gaap_ReclassificationOtherMember_36b1eeba-15a8-4e92-9ce8-c444491f808a" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EarningsPerShareAntidilutiveStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#EarningsPerShareAntidilutiveStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EarningsPerShareAntidilutiveStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails" xlink:type="extended" id="i6e5501a3bc3844eaa684b8f318ba9df7_EarningsPerShareAntidilutiveStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_9af4b487-92fd-4886-b169-3ae81f31dcda" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_83611d1f-8e84-430c-b950-34bd084065bd" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_9af4b487-92fd-4886-b169-3ae81f31dcda" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_83611d1f-8e84-430c-b950-34bd084065bd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01b0df66-8a15-4d3b-bf26-39c7036ee154" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_9af4b487-92fd-4886-b169-3ae81f31dcda" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01b0df66-8a15-4d3b-bf26-39c7036ee154" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_bcff7944-4e2f-43c3-b8bc-a03490a62687" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01b0df66-8a15-4d3b-bf26-39c7036ee154" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_bcff7944-4e2f-43c3-b8bc-a03490a62687" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_bcff7944-4e2f-43c3-b8bc-a03490a62687_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_bcff7944-4e2f-43c3-b8bc-a03490a62687" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_bcff7944-4e2f-43c3-b8bc-a03490a62687_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_f10188f4-57a1-4148-bf1d-3eb5cc47ce3f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_bcff7944-4e2f-43c3-b8bc-a03490a62687" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_f10188f4-57a1-4148-bf1d-3eb5cc47ce3f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_84429ffa-767c-45a8-ac68-f21b0e32a521" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_f10188f4-57a1-4148-bf1d-3eb5cc47ce3f" xlink:to="loc_us-gaap_EmployeeStockOptionMember_84429ffa-767c-45a8-ac68-f21b0e32a521" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EarningsPerShareNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#EarningsPerShareNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EarningsPerShareNarrativeDetails" xlink:type="extended" id="i9d0af28f63fe46069d2ab57821920323_EarningsPerShareNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_284fa508-9ed5-4a0e-9db1-1c457b3a275a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchaseProgramAuthorizedAmount1_aeb948b5-46c8-4038-ae08-1fd9c60cc7cc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockRepurchaseProgramAuthorizedAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_284fa508-9ed5-4a0e-9db1-1c457b3a275a" xlink:to="loc_us-gaap_StockRepurchaseProgramAuthorizedAmount1_aeb948b5-46c8-4038-ae08-1fd9c60cc7cc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1_af6d7b0c-f44d-464d-ad32-a801566ae162" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_284fa508-9ed5-4a0e-9db1-1c457b3a275a" xlink:to="loc_us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1_af6d7b0c-f44d-464d-ad32-a801566ae162" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CommonStockDividendRateIncreasePercentage_c19a5825-2b3c-4ff3-9b3b-fbd190ac2bda" xlink:href="csx-20220331.xsd#csx_CommonStockDividendRateIncreasePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_284fa508-9ed5-4a0e-9db1-1c457b3a275a" xlink:to="loc_csx_CommonStockDividendRateIncreasePercentage_c19a5825-2b3c-4ff3-9b3b-fbd190ac2bda" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockDividendsPerShareCashPaid_f144ce37-8aef-4bd9-88d1-02dffbf2073f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommonStockDividendsPerShareCashPaid"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_284fa508-9ed5-4a0e-9db1-1c457b3a275a" xlink:to="loc_us-gaap_CommonStockDividendsPerShareCashPaid_f144ce37-8aef-4bd9-88d1-02dffbf2073f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_ca24a6bd-8c72-4922-8ef6-e07e6c161b5b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_284fa508-9ed5-4a0e-9db1-1c457b3a275a" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_ca24a6bd-8c72-4922-8ef6-e07e6c161b5b" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareRepurchaseProgramAxis_ee889ee9-1c90-49f6-b96a-49d1d9a8f3de" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareRepurchaseProgramAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_ca24a6bd-8c72-4922-8ef6-e07e6c161b5b" xlink:to="loc_us-gaap_ShareRepurchaseProgramAxis_ee889ee9-1c90-49f6-b96a-49d1d9a8f3de" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareRepurchaseProgramDomain_ee889ee9-1c90-49f6-b96a-49d1d9a8f3de_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareRepurchaseProgramDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ShareRepurchaseProgramAxis_ee889ee9-1c90-49f6-b96a-49d1d9a8f3de" xlink:to="loc_us-gaap_ShareRepurchaseProgramDomain_ee889ee9-1c90-49f6-b96a-49d1d9a8f3de_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareRepurchaseProgramDomain_98064700-0bef-40f3-9606-cc0b847b6e19" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareRepurchaseProgramDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ShareRepurchaseProgramAxis_ee889ee9-1c90-49f6-b96a-49d1d9a8f3de" xlink:to="loc_us-gaap_ShareRepurchaseProgramDomain_98064700-0bef-40f3-9606-cc0b847b6e19" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareRepurchaseProgramJanuary2019Member_c0bcb960-22fa-46e6-913b-70d6a4d27848" xlink:href="csx-20220331.xsd#csx_ShareRepurchaseProgramJanuary2019Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareRepurchaseProgramDomain_98064700-0bef-40f3-9606-cc0b847b6e19" xlink:to="loc_csx_ShareRepurchaseProgramJanuary2019Member_c0bcb960-22fa-46e6-913b-70d6a4d27848" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareRepurchaseProgramOctober2020Member_94efe13b-c815-457b-89e5-36c843da3629" xlink:href="csx-20220331.xsd#csx_ShareRepurchaseProgramOctober2020Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareRepurchaseProgramDomain_98064700-0bef-40f3-9606-cc0b847b6e19" xlink:to="loc_csx_ShareRepurchaseProgramOctober2020Member_94efe13b-c815-457b-89e5-36c843da3629" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationPretaxExpenseandIncomeTaxBenefitsDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#StockPlansandShareBasedCompensationPretaxExpenseandIncomeTaxBenefitsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationPretaxExpenseandIncomeTaxBenefitsDetails" xlink:type="extended" id="i324fe76bcfc44ce1b40a3c56e2140d27_StockPlansandShareBasedCompensationPretaxExpenseandIncomeTaxBenefitsDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_444071fa-5add-47f2-932a-ef230836cf88" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_5c200eb0-0915-46df-a83f-dd21258188d9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_444071fa-5add-47f2-932a-ef230836cf88" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_5c200eb0-0915-46df-a83f-dd21258188d9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationTaxBenefitFromCompensationExpense_6f1edae0-748f-48bf-b570-c9905358557d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationTaxBenefitFromCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_444071fa-5add-47f2-932a-ef230836cf88" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationTaxBenefitFromCompensationExpense_6f1edae0-748f-48bf-b570-c9905358557d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_52aae701-dfcb-4f8d-afd6-1d47778377ec" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_444071fa-5add-47f2-932a-ef230836cf88" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_52aae701-dfcb-4f8d-afd6-1d47778377ec" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_3a94d306-7330-4e09-9e0a-6fae4e01df01" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_52aae701-dfcb-4f8d-afd6-1d47778377ec" xlink:to="loc_us-gaap_AwardTypeAxis_3a94d306-7330-4e09-9e0a-6fae4e01df01" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_3a94d306-7330-4e09-9e0a-6fae4e01df01_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_3a94d306-7330-4e09-9e0a-6fae4e01df01" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_3a94d306-7330-4e09-9e0a-6fae4e01df01_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_3a94d306-7330-4e09-9e0a-6fae4e01df01" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PerformanceSharesMember_9d52947f-d8ab-4804-91ee-183407fc27bb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PerformanceSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:to="loc_us-gaap_PerformanceSharesMember_9d52947f-d8ab-4804-91ee-183407fc27bb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_b23f170f-6121-48a6-b042-2a1ffd5736ef" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:to="loc_us-gaap_EmployeeStockOptionMember_b23f170f-6121-48a6-b042-2a1ffd5736ef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_bf4e2c72-33d0-48d8-a76c-62f14fdc96a6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_bf4e2c72-33d0-48d8-a76c-62f14fdc96a6" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_StockAwardsDirectorsMember_e6b23575-6645-434e-83b1-1fda775085f2" xlink:href="csx-20220331.xsd#csx_StockAwardsDirectorsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:to="loc_csx_StockAwardsDirectorsMember_e6b23575-6645-434e-83b1-1fda775085f2" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_56274d96-ab34-4cfd-9c63-d1cb6a409be8" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0619c823-f638-4d33-9b63-cfceb4543465" xlink:to="loc_us-gaap_EmployeeStockMember_56274d96-ab34-4cfd-9c63-d1cb6a409be8" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#StockPlansandShareBasedCompensationNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationNarrativeDetails" xlink:type="extended" id="i0f020e48165f41eabcbf93ed2dd806e5_StockPlansandShareBasedCompensationNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_514a9bd0-c727-454e-a5b9-a4007f630ab2" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_514a9bd0-c727-454e-a5b9-a4007f630ab2" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_PercentageOfGrantsWithPerformanceVestingAverageAnnualOperatingIncomeGrowth_d017d9c8-8eab-4bd4-8428-17e236ee0251" xlink:href="csx-20220331.xsd#csx_PercentageOfGrantsWithPerformanceVestingAverageAnnualOperatingIncomeGrowth"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_PercentageOfGrantsWithPerformanceVestingAverageAnnualOperatingIncomeGrowth_d017d9c8-8eab-4bd4-8428-17e236ee0251" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_PercentageOfGrantsWithPerformanceVestingCumulativeFreeCashFlow_572c6f6d-af42-41f0-be81-15d9d1825970" xlink:href="csx-20220331.xsd#csx_PercentageOfGrantsWithPerformanceVestingCumulativeFreeCashFlow"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_PercentageOfGrantsWithPerformanceVestingCumulativeFreeCashFlow_572c6f6d-af42-41f0-be81-15d9d1825970" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfEquivalentSharesOfCommonStockPerUnit_8cfe9128-6fff-48dc-bcaf-a62c2a40e949" xlink:href="csx-20220331.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfEquivalentSharesOfCommonStockPerUnit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfEquivalentSharesOfCommonStockPerUnit_8cfe9128-6fff-48dc-bcaf-a62c2a40e949" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SharebasedCompensationArrangementbySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageofTarget_50db5630-d989-4e72-9a4e-dd2417da3834" xlink:href="csx-20220331.xsd#csx_SharebasedCompensationArrangementbySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageofTarget"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_SharebasedCompensationArrangementbySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageofTarget_50db5630-d989-4e72-9a4e-dd2417da3834" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageAdjustment_adeb13c7-229a-491e-be9e-e03e08a00c52" xlink:href="csx-20220331.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageAdjustment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageAdjustment_adeb13c7-229a-491e-be9e-e03e08a00c52" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_de60f1f1-e53b-40dc-ac27-38cf509e2cc2" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_de60f1f1-e53b-40dc-ac27-38cf509e2cc2" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_b2403627-afa7-4e65-8fe9-618b5b55c244" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_b2403627-afa7-4e65-8fe9-618b5b55c244" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_35ad6fd2-396c-4253-8e9e-3fe751a2fb33" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_35ad6fd2-396c-4253-8e9e-3fe751a2fb33" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_70f0911d-ca44-43ba-8e50-7cb3b7fc039f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_70f0911d-ca44-43ba-8e50-7cb3b7fc039f" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SharebasedCompensationArrangementbySharebasedPaymentAwardMinimumEmployeeSubscriptionRate_b4830ea6-a4d1-4e4c-a927-11174aa39cfe" xlink:href="csx-20220331.xsd#csx_SharebasedCompensationArrangementbySharebasedPaymentAwardMinimumEmployeeSubscriptionRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_SharebasedCompensationArrangementbySharebasedPaymentAwardMinimumEmployeeSubscriptionRate_b4830ea6-a4d1-4e4c-a927-11174aa39cfe" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumEmployeeSubscriptionRate_fea2a1c4-1ba2-4073-9143-ae61e908453d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumEmployeeSubscriptionRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumEmployeeSubscriptionRate_fea2a1c4-1ba2-4073-9143-ae61e908453d" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumStockValuePurchasedPerEmployee_dd24f07d-6386-49ba-910b-aff1a44ffca3" xlink:href="csx-20220331.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumStockValuePurchasedPerEmployee"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumStockValuePurchasedPerEmployee_dd24f07d-6386-49ba-910b-aff1a44ffca3" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent_a5a72b23-0c05-4aad-9f3b-25d60a73f660" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent_a5a72b23-0c05-4aad-9f3b-25d60a73f660" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SharebasedCompensationArrangementbySharebasedPaymentAwardShareOfferingPeriod_256b8aa0-20cd-4652-bd9e-3b5f2ea90d74" xlink:href="csx-20220331.xsd#csx_SharebasedCompensationArrangementbySharebasedPaymentAwardShareOfferingPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_csx_SharebasedCompensationArrangementbySharebasedPaymentAwardShareOfferingPeriod_256b8aa0-20cd-4652-bd9e-3b5f2ea90d74" xlink:type="arc" order="14"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_0735eff7-a442-49f1-b47a-47990554cb13" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_a55678fb-fe40-4870-ad23-ea7b4e0a6913" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_0735eff7-a442-49f1-b47a-47990554cb13" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_fbfbc3a2-45bd-499a-8bd7-6beabf86d9d9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_0735eff7-a442-49f1-b47a-47990554cb13" xlink:to="loc_us-gaap_PlanNameAxis_fbfbc3a2-45bd-499a-8bd7-6beabf86d9d9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_fbfbc3a2-45bd-499a-8bd7-6beabf86d9d9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_fbfbc3a2-45bd-499a-8bd7-6beabf86d9d9" xlink:to="loc_us-gaap_PlanNameDomain_fbfbc3a2-45bd-499a-8bd7-6beabf86d9d9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_7e6390fd-80d0-4870-a5e2-7eb1ffbd5d83" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_fbfbc3a2-45bd-499a-8bd7-6beabf86d9d9" xlink:to="loc_us-gaap_PlanNameDomain_7e6390fd-80d0-4870-a5e2-7eb1ffbd5d83" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongtermIncentivePlanMember_508049ed-ac6a-49d0-9f9b-92abd81b4ea4" xlink:href="csx-20220331.xsd#csx_LongtermIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_7e6390fd-80d0-4870-a5e2-7eb1ffbd5d83" xlink:to="loc_csx_LongtermIncentivePlanMember_508049ed-ac6a-49d0-9f9b-92abd81b4ea4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_EmployeeStockPurchasePlanMember_2669d21f-673f-4ff9-a4d0-b5b2893474ea" xlink:href="csx-20220331.xsd#csx_EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_7e6390fd-80d0-4870-a5e2-7eb1ffbd5d83" xlink:to="loc_csx_EmployeeStockPurchasePlanMember_2669d21f-673f-4ff9-a4d0-b5b2893474ea" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_f70a1d9b-7ed3-4817-9c98-7777f84c6d8f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_0735eff7-a442-49f1-b47a-47990554cb13" xlink:to="loc_us-gaap_AwardTypeAxis_f70a1d9b-7ed3-4817-9c98-7777f84c6d8f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_f70a1d9b-7ed3-4817-9c98-7777f84c6d8f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_f70a1d9b-7ed3-4817-9c98-7777f84c6d8f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_f70a1d9b-7ed3-4817-9c98-7777f84c6d8f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9662d569-6850-48f7-99af-83cb2fea8121" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_f70a1d9b-7ed3-4817-9c98-7777f84c6d8f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9662d569-6850-48f7-99af-83cb2fea8121" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PerformanceSharesMember_e1e95da3-3d07-4f53-bb17-c82851f65e5f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PerformanceSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9662d569-6850-48f7-99af-83cb2fea8121" xlink:to="loc_us-gaap_PerformanceSharesMember_e1e95da3-3d07-4f53-bb17-c82851f65e5f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_cf83b7f9-9363-4400-a4c9-194ea2f754ff" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9662d569-6850-48f7-99af-83cb2fea8121" xlink:to="loc_us-gaap_EmployeeStockOptionMember_cf83b7f9-9363-4400-a4c9-194ea2f754ff" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_78806632-4b92-4d9d-a1cc-5042dbf079b6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9662d569-6850-48f7-99af-83cb2fea8121" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_78806632-4b92-4d9d-a1cc-5042dbf079b6" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_30093f9e-7ec7-46e6-8e20-ea2a83c5a263" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9662d569-6850-48f7-99af-83cb2fea8121" xlink:to="loc_us-gaap_EmployeeStockMember_30093f9e-7ec7-46e6-8e20-ea2a83c5a263" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_TitleOfIndividualAxis_f7dca8b1-3bf4-49f4-ba2c-fcee26b0aa6b" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_TitleOfIndividualAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_0735eff7-a442-49f1-b47a-47990554cb13" xlink:to="loc_srt_TitleOfIndividualAxis_f7dca8b1-3bf4-49f4-ba2c-fcee26b0aa6b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_f7dca8b1-3bf4-49f4-ba2c-fcee26b0aa6b_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_TitleOfIndividualAxis_f7dca8b1-3bf4-49f4-ba2c-fcee26b0aa6b" xlink:to="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_f7dca8b1-3bf4-49f4-ba2c-fcee26b0aa6b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_f77d97d6-ba27-41aa-b771-c70e4fab4522" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_TitleOfIndividualAxis_f7dca8b1-3bf4-49f4-ba2c-fcee26b0aa6b" xlink:to="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_f77d97d6-ba27-41aa-b771-c70e4fab4522" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CertainEmployeesMember_0dbdd896-37c5-4c35-8890-4c18fd476f61" xlink:href="csx-20220331.xsd#csx_CertainEmployeesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_f77d97d6-ba27-41aa-b771-c70e4fab4522" xlink:to="loc_csx_CertainEmployeesMember_0dbdd896-37c5-4c35-8890-4c18fd476f61" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ExecutiveOfficerMember_5cd869dd-b1c9-4a75-8cb7-22c588eda3f8" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_ExecutiveOfficerMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_f77d97d6-ba27-41aa-b771-c70e4fab4522" xlink:to="loc_srt_ExecutiveOfficerMember_5cd869dd-b1c9-4a75-8cb7-22c588eda3f8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_56d9eba1-1bf0-4870-936d-0c6f01911263" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_0735eff7-a442-49f1-b47a-47990554cb13" xlink:to="loc_srt_RangeAxis_56d9eba1-1bf0-4870-936d-0c6f01911263" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_56d9eba1-1bf0-4870-936d-0c6f01911263_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_56d9eba1-1bf0-4870-936d-0c6f01911263" xlink:to="loc_srt_RangeMember_56d9eba1-1bf0-4870-936d-0c6f01911263_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_a5887fbd-c647-4cef-a7ca-98be59c2a7d4" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_56d9eba1-1bf0-4870-936d-0c6f01911263" xlink:to="loc_srt_RangeMember_a5887fbd-c647-4cef-a7ca-98be59c2a7d4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_802249ae-481f-4723-82af-8f1dbd2ce043" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_a5887fbd-c647-4cef-a7ca-98be59c2a7d4" xlink:to="loc_srt_MinimumMember_802249ae-481f-4723-82af-8f1dbd2ce043" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_894d3045-edab-4d0f-aa62-2033a143e7fe" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_a5887fbd-c647-4cef-a7ca-98be59c2a7d4" xlink:to="loc_srt_MaximumMember_894d3045-edab-4d0f-aa62-2033a143e7fe" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationWeightedAverageAssumptionsUsedforPerformanceUnitsDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#StockPlansandShareBasedCompensationWeightedAverageAssumptionsUsedforPerformanceUnitsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationWeightedAverageAssumptionsUsedforPerformanceUnitsDetails" xlink:type="extended" id="i4fa8e0b75c7448b29759bf6badae5319_StockPlansandShareBasedCompensationWeightedAverageAssumptionsUsedforPerformanceUnitsDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_7c8b8631-02bc-401b-bf65-fe0a9b022b4e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_f033b6e0-ec42-4f86-9ac1-9c0f3e18a636" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_7c8b8631-02bc-401b-bf65-fe0a9b022b4e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_f033b6e0-ec42-4f86-9ac1-9c0f3e18a636" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_74ebcd12-28ae-4df6-a166-0223c8b7eb48" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_f033b6e0-ec42-4f86-9ac1-9c0f3e18a636" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_74ebcd12-28ae-4df6-a166-0223c8b7eb48" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_f0d54ddf-df25-4b95-992b-f0cb066667ad" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_f033b6e0-ec42-4f86-9ac1-9c0f3e18a636" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_f0d54ddf-df25-4b95-992b-f0cb066667ad" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_7e5e0f4d-999b-47c6-afd3-c97dd7ccf290" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_f033b6e0-ec42-4f86-9ac1-9c0f3e18a636" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_7e5e0f4d-999b-47c6-afd3-c97dd7ccf290" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_78dd0ec8-71b1-4064-b99f-5beda67d378f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_7c8b8631-02bc-401b-bf65-fe0a9b022b4e" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_78dd0ec8-71b1-4064-b99f-5beda67d378f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_2803376d-a0fc-4173-b333-c1d69299d1fb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_78dd0ec8-71b1-4064-b99f-5beda67d378f" xlink:to="loc_us-gaap_AwardTypeAxis_2803376d-a0fc-4173-b333-c1d69299d1fb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_2803376d-a0fc-4173-b333-c1d69299d1fb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_2803376d-a0fc-4173-b333-c1d69299d1fb" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_2803376d-a0fc-4173-b333-c1d69299d1fb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_09e33088-56bd-4ba9-9ba7-e29afe92b46a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_2803376d-a0fc-4173-b333-c1d69299d1fb" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_09e33088-56bd-4ba9-9ba7-e29afe92b46a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PerformanceSharesMember_cc39aebe-0f07-4606-9119-c91f0485233f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PerformanceSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_09e33088-56bd-4ba9-9ba7-e29afe92b46a" xlink:to="loc_us-gaap_PerformanceSharesMember_cc39aebe-0f07-4606-9119-c91f0485233f" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationFairValueAssumptionsUsedforStockOptionsDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#StockPlansandShareBasedCompensationFairValueAssumptionsUsedforStockOptionsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationFairValueAssumptionsUsedforStockOptionsDetails" xlink:type="extended" id="ia02d459d9bd2497882583fe8968380b6_StockPlansandShareBasedCompensationFairValueAssumptionsUsedforStockOptionsDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_3f042579-07d8-4f7c-9a37-b7de468f4bd0" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_05dff145-1b6e-4cab-9261-79a13bd21e58" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_3f042579-07d8-4f7c-9a37-b7de468f4bd0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_05dff145-1b6e-4cab-9261-79a13bd21e58" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_d774cb2e-96a4-4e76-afc9-3a97fb4efe82" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_3f042579-07d8-4f7c-9a37-b7de468f4bd0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_d774cb2e-96a4-4e76-afc9-3a97fb4efe82" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_ad3db045-7738-4b3a-b277-82580611f51c" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_d774cb2e-96a4-4e76-afc9-3a97fb4efe82" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_ad3db045-7738-4b3a-b277-82580611f51c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_95d5a0ec-9df0-4fe2-8c55-d0b01f6f638f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_d774cb2e-96a4-4e76-afc9-3a97fb4efe82" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_95d5a0ec-9df0-4fe2-8c55-d0b01f6f638f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_26b41e4f-365d-4ced-92ed-cb4e44b80914" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_d774cb2e-96a4-4e76-afc9-3a97fb4efe82" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_26b41e4f-365d-4ced-92ed-cb4e44b80914" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_15f6f09e-6bb0-4180-b3be-e6d2aac730ed" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract_d774cb2e-96a4-4e76-afc9-3a97fb4efe82" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_15f6f09e-6bb0-4180-b3be-e6d2aac730ed" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_4cd88f03-1d58-4c2e-ab8d-c331568fb428" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_3f042579-07d8-4f7c-9a37-b7de468f4bd0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_4cd88f03-1d58-4c2e-ab8d-c331568fb428" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_58435ee6-83b4-4fce-83d0-cf9d4adcf93f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_4cd88f03-1d58-4c2e-ab8d-c331568fb428" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_58435ee6-83b4-4fce-83d0-cf9d4adcf93f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_cabd2077-c00c-4ff8-9da1-af647302901b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_3f042579-07d8-4f7c-9a37-b7de468f4bd0" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_cabd2077-c00c-4ff8-9da1-af647302901b" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_c474dacd-46cf-4221-9581-a90e36695d6f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_cabd2077-c00c-4ff8-9da1-af647302901b" xlink:to="loc_us-gaap_AwardTypeAxis_c474dacd-46cf-4221-9581-a90e36695d6f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_c474dacd-46cf-4221-9581-a90e36695d6f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_c474dacd-46cf-4221-9581-a90e36695d6f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_c474dacd-46cf-4221-9581-a90e36695d6f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_85c64153-eec0-407b-b605-690537cfec58" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_c474dacd-46cf-4221-9581-a90e36695d6f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_85c64153-eec0-407b-b605-690537cfec58" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_25bab173-a41c-4558-824e-dbe6b11612d9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_85c64153-eec0-407b-b605-690537cfec58" xlink:to="loc_us-gaap_EmployeeStockOptionMember_25bab173-a41c-4558-824e-dbe6b11612d9" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationSharesIssuedUnderEmployeeStockPurchasePlanDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#StockPlansandShareBasedCompensationSharesIssuedUnderEmployeeStockPurchasePlanDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationSharesIssuedUnderEmployeeStockPurchasePlanDetails" xlink:type="extended" id="i00f10f1fc31d423db7a155267bf452f5_StockPlansandShareBasedCompensationSharesIssuedUnderEmployeeStockPurchasePlanDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_61aa6523-8708-43c3-a6fb-a43668479d9c" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesIssuedInPeriod_43dd6d6f-9f44-46fc-81e2-c302735d70cd" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesIssuedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_61aa6523-8708-43c3-a6fb-a43668479d9c" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesIssuedInPeriod_43dd6d6f-9f44-46fc-81e2-c302735d70cd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardPerShareWeightedAveragePriceOfSharesIssued_5965aa27-51c8-4605-997a-a030a4155bb8" xlink:href="csx-20220331.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardPerShareWeightedAveragePriceOfSharesIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_61aa6523-8708-43c3-a6fb-a43668479d9c" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardPerShareWeightedAveragePriceOfSharesIssued_5965aa27-51c8-4605-997a-a030a4155bb8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_fc6e3719-d57a-4238-a430-fd76772e1195" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_61aa6523-8708-43c3-a6fb-a43668479d9c" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_fc6e3719-d57a-4238-a430-fd76772e1195" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_3dfc87e6-957f-406a-a2e4-136de3d93a64" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_fc6e3719-d57a-4238-a430-fd76772e1195" xlink:to="loc_us-gaap_PlanNameAxis_3dfc87e6-957f-406a-a2e4-136de3d93a64" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_3dfc87e6-957f-406a-a2e4-136de3d93a64_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_3dfc87e6-957f-406a-a2e4-136de3d93a64" xlink:to="loc_us-gaap_PlanNameDomain_3dfc87e6-957f-406a-a2e4-136de3d93a64_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_d8f62037-2cbf-47ef-a744-059c6d493bf6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_3dfc87e6-957f-406a-a2e4-136de3d93a64" xlink:to="loc_us-gaap_PlanNameDomain_d8f62037-2cbf-47ef-a744-059c6d493bf6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_EmployeeStockPurchasePlanMember_85161316-5938-4893-8d86-9efdc2d3808e" xlink:href="csx-20220331.xsd#csx_EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_d8f62037-2cbf-47ef-a744-059c6d493bf6" xlink:to="loc_csx_EmployeeStockPurchasePlanMember_85161316-5938-4893-8d86-9efdc2d3808e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_6b7d51c6-74e7-4458-a1f9-34d78ee9b2fa" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_fc6e3719-d57a-4238-a430-fd76772e1195" xlink:to="loc_us-gaap_AwardTypeAxis_6b7d51c6-74e7-4458-a1f9-34d78ee9b2fa" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_6b7d51c6-74e7-4458-a1f9-34d78ee9b2fa_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_6b7d51c6-74e7-4458-a1f9-34d78ee9b2fa" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_6b7d51c6-74e7-4458-a1f9-34d78ee9b2fa_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_864c5cdc-3ba0-4eeb-a5c8-bf6d40fba5ad" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_6b7d51c6-74e7-4458-a1f9-34d78ee9b2fa" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_864c5cdc-3ba0-4eeb-a5c8-bf6d40fba5ad" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_baff42e0-ffc5-491d-bb13-df65615282c2" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_864c5cdc-3ba0-4eeb-a5c8-bf6d40fba5ad" xlink:to="loc_us-gaap_EmployeeStockMember_baff42e0-ffc5-491d-bb13-df65615282c2" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesScheduleofCasualtyEnvironmentalandOtherReservesDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#CasualtyEnvironmentalandOtherReservesScheduleofCasualtyEnvironmentalandOtherReservesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesScheduleofCasualtyEnvironmentalandOtherReservesDetails" xlink:type="extended" id="i5b09e720ce7b4b23af014a2d5dcd5c91_CasualtyEnvironmentalandOtherReservesScheduleofCasualtyEnvironmentalandOtherReservesDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_b2b3d684-953d-4182-baec-7c6d0efcd760" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualCarryingValueCurrent_19aa94ea-01ba-4b7d-b8f4-47c53bcf6dea" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyAccrualCarryingValueCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_b2b3d684-953d-4182-baec-7c6d0efcd760" xlink:to="loc_us-gaap_LossContingencyAccrualCarryingValueCurrent_19aa94ea-01ba-4b7d-b8f4-47c53bcf6dea" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualCarryingValueNoncurrent_a937ba5b-e8f6-4397-9e22-120626de4ac3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyAccrualCarryingValueNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_b2b3d684-953d-4182-baec-7c6d0efcd760" xlink:to="loc_us-gaap_LossContingencyAccrualCarryingValueNoncurrent_a937ba5b-e8f6-4397-9e22-120626de4ac3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualAtCarryingValue_c36d1bdc-91f5-43d3-9e64-a82f71246eca" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyAccrualAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_b2b3d684-953d-4182-baec-7c6d0efcd760" xlink:to="loc_us-gaap_LossContingencyAccrualAtCarryingValue_c36d1bdc-91f5-43d3-9e64-a82f71246eca" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesTable_fda312cc-2ccf-4a5a-b383-f56fd6f7a7b9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_b2b3d684-953d-4182-baec-7c6d0efcd760" xlink:to="loc_us-gaap_LossContingenciesTable_fda312cc-2ccf-4a5a-b383-f56fd6f7a7b9" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_bcd11888-e892-4f0e-8bf1-1e5ba24c9fc2" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_fda312cc-2ccf-4a5a-b383-f56fd6f7a7b9" xlink:to="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_bcd11888-e892-4f0e-8bf1-1e5ba24c9fc2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_bcd11888-e892-4f0e-8bf1-1e5ba24c9fc2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_bcd11888-e892-4f0e-8bf1-1e5ba24c9fc2" xlink:to="loc_us-gaap_LossContingencyNatureDomain_bcd11888-e892-4f0e-8bf1-1e5ba24c9fc2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_9e23ed7f-d1b7-4fb4-969d-32fe2cc18319" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_bcd11888-e892-4f0e-8bf1-1e5ba24c9fc2" xlink:to="loc_us-gaap_LossContingencyNatureDomain_9e23ed7f-d1b7-4fb4-969d-32fe2cc18319" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalCasualtyMember_b4c83ee9-2a7b-47d5-9af4-bff6872de8f1" xlink:href="csx-20220331.xsd#csx_TotalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_9e23ed7f-d1b7-4fb4-969d-32fe2cc18319" xlink:to="loc_csx_TotalCasualtyMember_b4c83ee9-2a7b-47d5-9af4-bff6872de8f1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_PersonalInjuryCasualtyMember_7ff417d2-75b2-4023-b675-957855d97f13" xlink:href="csx-20220331.xsd#csx_PersonalInjuryCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalCasualtyMember_b4c83ee9-2a7b-47d5-9af4-bff6872de8f1" xlink:to="loc_csx_PersonalInjuryCasualtyMember_7ff417d2-75b2-4023-b675-957855d97f13" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OccupationalCasualtyMember_48308764-8016-4296-997c-13488fea4f41" xlink:href="csx-20220331.xsd#csx_OccupationalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalCasualtyMember_b4c83ee9-2a7b-47d5-9af4-bff6872de8f1" xlink:to="loc_csx_OccupationalCasualtyMember_48308764-8016-4296-997c-13488fea4f41" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EnvironmentalIssueMember_50078752-1c20-4279-9150-f91053c7a690" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EnvironmentalIssueMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_9e23ed7f-d1b7-4fb4-969d-32fe2cc18319" xlink:to="loc_us-gaap_EnvironmentalIssueMember_50078752-1c20-4279-9150-f91053c7a690" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OtherReservesMember_ed2991df-85c0-48a4-8131-55458e720bb7" xlink:href="csx-20220331.xsd#csx_OtherReservesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_9e23ed7f-d1b7-4fb4-969d-32fe2cc18319" xlink:to="loc_csx_OtherReservesMember_ed2991df-85c0-48a4-8131-55458e720bb7" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#CasualtyEnvironmentalandOtherReservesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesNarrativeDetails" xlink:type="extended" id="ie20d8e4010324825b3f439bdfb37a61b_CasualtyEnvironmentalandOtherReservesNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_1c1b8be3-617e-4d09-8597-302778ad9a6d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualAtCarryingValue_ffbd9532-b9b3-4f88-ace5-b407024a873e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyAccrualAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_1c1b8be3-617e-4d09-8597-302778ad9a6d" xlink:to="loc_us-gaap_LossContingencyAccrualAtCarryingValue_ffbd9532-b9b3-4f88-ace5-b407024a873e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SelfInsuredRetention_6c403cfb-daaa-482d-a3ee-d4d8a1668bff" xlink:href="csx-20220331.xsd#csx_SelfInsuredRetention"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_1c1b8be3-617e-4d09-8597-302778ad9a6d" xlink:to="loc_csx_SelfInsuredRetention_6c403cfb-daaa-482d-a3ee-d4d8a1668bff" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyNumberOfClaimsExpectedToExceedSelfInsuredRetentionAmount_bc6b29bc-69b4-45eb-b7b4-b245d7cc63b8" xlink:href="csx-20220331.xsd#csx_LossContingencyNumberOfClaimsExpectedToExceedSelfInsuredRetentionAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_1c1b8be3-617e-4d09-8597-302778ad9a6d" xlink:to="loc_csx_LossContingencyNumberOfClaimsExpectedToExceedSelfInsuredRetentionAmount_bc6b29bc-69b4-45eb-b7b4-b245d7cc63b8" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfImpairedSites_7c33f9d0-9ec8-493f-9ad3-fd9707ae348c" xlink:href="csx-20220331.xsd#csx_NumberOfImpairedSites"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_1c1b8be3-617e-4d09-8597-302778ad9a6d" xlink:to="loc_csx_NumberOfImpairedSites_7c33f9d0-9ec8-493f-9ad3-fd9707ae348c" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesTable_acd5df73-9010-4896-8f98-7a9f1f2f39b3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_1c1b8be3-617e-4d09-8597-302778ad9a6d" xlink:to="loc_us-gaap_LossContingenciesTable_acd5df73-9010-4896-8f98-7a9f1f2f39b3" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_e3aa890c-1d2e-4afc-9370-434f0f38ecfe" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_acd5df73-9010-4896-8f98-7a9f1f2f39b3" xlink:to="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_e3aa890c-1d2e-4afc-9370-434f0f38ecfe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_e3aa890c-1d2e-4afc-9370-434f0f38ecfe_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_e3aa890c-1d2e-4afc-9370-434f0f38ecfe" xlink:to="loc_us-gaap_LossContingencyNatureDomain_e3aa890c-1d2e-4afc-9370-434f0f38ecfe_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_e597307b-f85a-4462-9cab-b0ca606c7df6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_e3aa890c-1d2e-4afc-9370-434f0f38ecfe" xlink:to="loc_us-gaap_LossContingencyNatureDomain_e597307b-f85a-4462-9cab-b0ca606c7df6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalCasualtyMember_97e0e831-3a40-400d-87b9-710730cc6ba2" xlink:href="csx-20220331.xsd#csx_TotalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_e597307b-f85a-4462-9cab-b0ca606c7df6" xlink:to="loc_csx_TotalCasualtyMember_97e0e831-3a40-400d-87b9-710730cc6ba2" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EnvironmentalIssueMember_abf65542-554c-4090-95c2-c3a5abc23283" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EnvironmentalIssueMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_e597307b-f85a-4462-9cab-b0ca606c7df6" xlink:to="loc_us-gaap_EnvironmentalIssueMember_abf65542-554c-4090-95c2-c3a5abc23283" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OtherReservesMember_59454e30-7006-4ce6-96e2-708dc49c0b5c" xlink:href="csx-20220331.xsd#csx_OtherReservesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_e597307b-f85a-4462-9cab-b0ca606c7df6" xlink:to="loc_csx_OtherReservesMember_59454e30-7006-4ce6-96e2-708dc49c0b5c" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CommitmentsandContingenciesDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#CommitmentsandContingenciesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CommitmentsandContingenciesDetails" xlink:type="extended" id="i63a847db493c4549a7d7fc9e1715e5b4_CommitmentsandContingenciesDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CasualtyAndCatastrophicPropertyDeductible_6cbd7126-d0a3-47e0-a36d-5f142ae0124a" xlink:href="csx-20220331.xsd#csx_CasualtyAndCatastrophicPropertyDeductible"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_CasualtyAndCatastrophicPropertyDeductible_6cbd7126-d0a3-47e0-a36d-5f142ae0124a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CasualtyAndNonCatastrophicPropertyDeductible_0e06ed25-be31-492f-bdc0-cd6e925a5eab" xlink:href="csx-20220331.xsd#csx_CasualtyAndNonCatastrophicPropertyDeductible"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_CasualtyAndNonCatastrophicPropertyDeductible_0e06ed25-be31-492f-bdc0-cd6e925a5eab" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SelfInsuredRetention_2955405d-f25a-4338-ae2d-d0a95548003c" xlink:href="csx-20220331.xsd#csx_SelfInsuredRetention"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_SelfInsuredRetention_2955405d-f25a-4338-ae2d-d0a95548003c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyEstimateOfPossibleLoss_d6746da1-6ca4-495c-a5a0-a59e0be194fe" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyEstimateOfPossibleLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_us-gaap_LossContingencyEstimateOfPossibleLoss_d6746da1-6ca4-495c-a5a0-a59e0be194fe" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyClaimsFiledNumberOfEntities_46ed9602-0c37-4f26-a981-d3d5c7b4669a" xlink:href="csx-20220331.xsd#csx_LossContingencyClaimsFiledNumberOfEntities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_LossContingencyClaimsFiledNumberOfEntities_46ed9602-0c37-4f26-a981-d3d5c7b4669a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyNumberOfConsolidatedClassActionLawsuits_9f23098e-67f0-470d-8df0-9818ac292bc2" xlink:href="csx-20220331.xsd#csx_LossContingencyNumberOfConsolidatedClassActionLawsuits"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_LossContingencyNumberOfConsolidatedClassActionLawsuits_9f23098e-67f0-470d-8df0-9818ac292bc2" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfMilesPertainingToPassaicRiverTidalReachRequiredToBeStudiedByEPA_2cfb444d-154c-4af9-8d5c-e40d051030de" xlink:href="csx-20220331.xsd#csx_NumberOfMilesPertainingToPassaicRiverTidalReachRequiredToBeStudiedByEPA"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_NumberOfMilesPertainingToPassaicRiverTidalReachRequiredToBeStudiedByEPA_2cfb444d-154c-4af9-8d5c-e40d051030de" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SiteContingencyNumberOfMilesUnderStudy_9a1e002d-3c77-4250-8cba-de9b15e7441c" xlink:href="csx-20220331.xsd#csx_SiteContingencyNumberOfMilesUnderStudy"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_SiteContingencyNumberOfMilesUnderStudy_9a1e002d-3c77-4250-8cba-de9b15e7441c" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SiteContingencyNumberOfPartiesParticipatingInAllocationProcessUnderStudy_7846d9c6-6059-45b7-9819-8232adb2b7b5" xlink:href="csx-20220331.xsd#csx_SiteContingencyNumberOfPartiesParticipatingInAllocationProcessUnderStudy"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_csx_SiteContingencyNumberOfPartiesParticipatingInAllocationProcessUnderStudy_7846d9c6-6059-45b7-9819-8232adb2b7b5" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNumberOfDefendants_b9e1fcac-29ad-48a6-8ce3-f5f023574ccb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNumberOfDefendants"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_us-gaap_LossContingencyNumberOfDefendants_b9e1fcac-29ad-48a6-8ce3-f5f023574ccb" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesTable_e22fd607-2150-4fa1-907a-a135a9d7bea8" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_15cffb40-3520-4da3-85d6-55f228580b42" xlink:to="loc_us-gaap_LossContingenciesTable_e22fd607-2150-4fa1-907a-a135a9d7bea8" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_690f54a8-48c8-4bc2-9f54-bfe645be7199" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_e22fd607-2150-4fa1-907a-a135a9d7bea8" xlink:to="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_690f54a8-48c8-4bc2-9f54-bfe645be7199" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_690f54a8-48c8-4bc2-9f54-bfe645be7199_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_690f54a8-48c8-4bc2-9f54-bfe645be7199" xlink:to="loc_us-gaap_LossContingencyNatureDomain_690f54a8-48c8-4bc2-9f54-bfe645be7199_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_0f54160f-fe27-4b92-9f58-ee982b252d0b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_690f54a8-48c8-4bc2-9f54-bfe645be7199" xlink:to="loc_us-gaap_LossContingencyNatureDomain_0f54160f-fe27-4b92-9f58-ee982b252d0b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalCasualtyMember_14e34159-d3be-4c11-a988-7377af23820b" xlink:href="csx-20220331.xsd#csx_TotalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_0f54160f-fe27-4b92-9f58-ee982b252d0b" xlink:to="loc_csx_TotalCasualtyMember_14e34159-d3be-4c11-a988-7377af23820b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_523752c9-9301-49e6-a89e-34faaa628ec0" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_e22fd607-2150-4fa1-907a-a135a9d7bea8" xlink:to="loc_srt_RangeAxis_523752c9-9301-49e6-a89e-34faaa628ec0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_523752c9-9301-49e6-a89e-34faaa628ec0_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_523752c9-9301-49e6-a89e-34faaa628ec0" xlink:to="loc_srt_RangeMember_523752c9-9301-49e6-a89e-34faaa628ec0_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_ee30e81e-1f55-4ea5-9ca6-5c364b086cdd" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_523752c9-9301-49e6-a89e-34faaa628ec0" xlink:to="loc_srt_RangeMember_ee30e81e-1f55-4ea5-9ca6-5c364b086cdd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_5b711627-52a4-4bfc-9ec0-bdd0a4218e56" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_ee30e81e-1f55-4ea5-9ca6-5c364b086cdd" xlink:to="loc_srt_MinimumMember_5b711627-52a4-4bfc-9ec0-bdd0a4218e56" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_253c8129-634c-4d3e-80af-ea5b6a390322" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_ee30e81e-1f55-4ea5-9ca6-5c364b086cdd" xlink:to="loc_srt_MaximumMember_253c8129-634c-4d3e-80af-ea5b6a390322" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationStatusAxis_0bc1b5c7-b3bb-42f7-a629-a34b43d48a8b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LitigationStatusAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_e22fd607-2150-4fa1-907a-a135a9d7bea8" xlink:to="loc_us-gaap_LitigationStatusAxis_0bc1b5c7-b3bb-42f7-a629-a34b43d48a8b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationStatusDomain_0bc1b5c7-b3bb-42f7-a629-a34b43d48a8b_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LitigationStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LitigationStatusAxis_0bc1b5c7-b3bb-42f7-a629-a34b43d48a8b" xlink:to="loc_us-gaap_LitigationStatusDomain_0bc1b5c7-b3bb-42f7-a629-a34b43d48a8b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationStatusDomain_b7ae0cb9-00fd-4cf9-a55a-a7bf086eb870" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LitigationStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LitigationStatusAxis_0bc1b5c7-b3bb-42f7-a629-a34b43d48a8b" xlink:to="loc_us-gaap_LitigationStatusDomain_b7ae0cb9-00fd-4cf9-a55a-a7bf086eb870" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PendingLitigationMember_4a19b644-518c-4c09-9269-22df39585e2d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PendingLitigationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LitigationStatusDomain_b7ae0cb9-00fd-4cf9-a55a-a7bf086eb870" xlink:to="loc_us-gaap_PendingLitigationMember_4a19b644-518c-4c09-9269-22df39585e2d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_LitigationCaseAxis_fbebd5c5-c6f8-462d-9806-ab1a76c06ad6" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_LitigationCaseAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_e22fd607-2150-4fa1-907a-a135a9d7bea8" xlink:to="loc_srt_LitigationCaseAxis_fbebd5c5-c6f8-462d-9806-ab1a76c06ad6" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_LitigationCaseTypeDomain_fbebd5c5-c6f8-462d-9806-ab1a76c06ad6_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_LitigationCaseTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_LitigationCaseAxis_fbebd5c5-c6f8-462d-9806-ab1a76c06ad6" xlink:to="loc_srt_LitigationCaseTypeDomain_fbebd5c5-c6f8-462d-9806-ab1a76c06ad6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_LitigationCaseTypeDomain_09b6ab5e-0c18-46ed-b298-a8a57bee01eb" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_LitigationCaseTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_LitigationCaseAxis_fbebd5c5-c6f8-462d-9806-ab1a76c06ad6" xlink:to="loc_srt_LitigationCaseTypeDomain_09b6ab5e-0c18-46ed-b298-a8a57bee01eb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelSurchargeAntitrustLitigationMember_03849293-bf97-4889-a2ba-37fd779c63f7" xlink:href="csx-20220331.xsd#csx_FuelSurchargeAntitrustLitigationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_LitigationCaseTypeDomain_09b6ab5e-0c18-46ed-b298-a8a57bee01eb" xlink:to="loc_csx_FuelSurchargeAntitrustLitigationMember_03849293-bf97-4889-a2ba-37fd779c63f7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_EnvironmentalLitigationMember_0c4f989d-6be7-464c-9e69-e7d0be315a3e" xlink:href="csx-20220331.xsd#csx_EnvironmentalLitigationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_LitigationCaseTypeDomain_09b6ab5e-0c18-46ed-b298-a8a57bee01eb" xlink:to="loc_csx_EnvironmentalLitigationMember_0c4f989d-6be7-464c-9e69-e7d0be315a3e" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EmployeeBenefitPlansNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#EmployeeBenefitPlansNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EmployeeBenefitPlansNarrativeDetails" xlink:type="extended" id="i43a9d93b793f49ce9f146c4d0420dc84_EmployeeBenefitPlansNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_e8a888bb-e418-452d-ba59-2d41650c76da" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanDisclosureLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DefinedBenefitPlanPlanAmendmentEliminationOfBenefitsMaximumAge_b1016f5b-8a9b-4cc9-a4a9-d3d8435a1c87" xlink:href="csx-20220331.xsd#csx_DefinedBenefitPlanPlanAmendmentEliminationOfBenefitsMaximumAge"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_e8a888bb-e418-452d-ba59-2d41650c76da" xlink:to="loc_csx_DefinedBenefitPlanPlanAmendmentEliminationOfBenefitsMaximumAge_b1016f5b-8a9b-4cc9-a4a9-d3d8435a1c87" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DefinedBenefitPlanPlanAmendmentEliminationOfBenefitsMinimumAge_417092ab-82cb-4228-858e-e8582b0e7ef8" xlink:href="csx-20220331.xsd#csx_DefinedBenefitPlanPlanAmendmentEliminationOfBenefitsMinimumAge"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_e8a888bb-e418-452d-ba59-2d41650c76da" xlink:to="loc_csx_DefinedBenefitPlanPlanAmendmentEliminationOfBenefitsMinimumAge_417092ab-82cb-4228-858e-e8582b0e7ef8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanExpectedFutureEmployerContributionsRemainderOfFiscalYear_e4637848-aac6-40d5-b598-3c2f1044e1cb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanExpectedFutureEmployerContributionsRemainderOfFiscalYear"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_e8a888bb-e418-452d-ba59-2d41650c76da" xlink:to="loc_us-gaap_DefinedBenefitPlanExpectedFutureEmployerContributionsRemainderOfFiscalYear_e4637848-aac6-40d5-b598-3c2f1044e1cb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_bdb80335-ddda-4e55-a2e2-e99c47631589" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_e8a888bb-e418-452d-ba59-2d41650c76da" xlink:to="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_bdb80335-ddda-4e55-a2e2-e99c47631589" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeAxis_bbdd98d9-2e11-448c-a98d-aedf4c9f3b77" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetirementPlanTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_bdb80335-ddda-4e55-a2e2-e99c47631589" xlink:to="loc_us-gaap_RetirementPlanTypeAxis_bbdd98d9-2e11-448c-a98d-aedf4c9f3b77" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeDomain_bbdd98d9-2e11-448c-a98d-aedf4c9f3b77_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetirementPlanTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RetirementPlanTypeAxis_bbdd98d9-2e11-448c-a98d-aedf4c9f3b77" xlink:to="loc_us-gaap_RetirementPlanTypeDomain_bbdd98d9-2e11-448c-a98d-aedf4c9f3b77_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeDomain_a4e56443-87f5-4955-b0d4-709fa3e2f77f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetirementPlanTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RetirementPlanTypeAxis_bbdd98d9-2e11-448c-a98d-aedf4c9f3b77" xlink:to="loc_us-gaap_RetirementPlanTypeDomain_a4e56443-87f5-4955-b0d4-709fa3e2f77f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherPostretirementBenefitPlansDefinedBenefitMember_2d7d0a18-cab8-4897-b899-950e70fe9bde" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OtherPostretirementBenefitPlansDefinedBenefitMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RetirementPlanTypeDomain_a4e56443-87f5-4955-b0d4-709fa3e2f77f" xlink:to="loc_us-gaap_OtherPostretirementBenefitPlansDefinedBenefitMember_2d7d0a18-cab8-4897-b899-950e70fe9bde" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EmployeeBenefitPlansComponentsofNetPeriodicBenefitCostsDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#EmployeeBenefitPlansComponentsofNetPeriodicBenefitCostsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EmployeeBenefitPlansComponentsofNetPeriodicBenefitCostsDetails" xlink:type="extended" id="i9a428b0faed54a219b9ef4683760441b_EmployeeBenefitPlansComponentsofNetPeriodicBenefitCostsDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_ebf8e9a3-83dc-44bb-8af3-6b4ed5003534" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanDisclosureLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_ebf8e9a3-83dc-44bb-8af3-6b4ed5003534" xlink:to="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanServiceCost_0e7d0001-6072-4166-b871-971fc5228c2a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanServiceCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_DefinedBenefitPlanServiceCost_0e7d0001-6072-4166-b871-971fc5228c2a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanInterestCost_801777c3-43c8-4612-9f66-03a967e009e2" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanInterestCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_DefinedBenefitPlanInterestCost_801777c3-43c8-4612-9f66-03a967e009e2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets_f0787513-e18c-4a6f-a331-e8988fa004e7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets_f0787513-e18c-4a6f-a331-e8988fa004e7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses_11087353-98a2-44c8-8eae-046ccd79e447" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses_11087353-98a2-44c8-8eae-046ccd79e447" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanAmortizationOfPriorServiceCostCredit_8ab028aa-b04d-4bc4-9313-ea863580e45d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanAmortizationOfPriorServiceCostCredit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_DefinedBenefitPlanAmortizationOfPriorServiceCostCredit_8ab028aa-b04d-4bc4-9313-ea863580e45d" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetPeriodicDefinedBenefitsExpenseReversalOfExpenseExcludingServiceCostComponent_b91848b5-c3bf-4601-b0e7-3954cfcf4885" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_NetPeriodicDefinedBenefitsExpenseReversalOfExpenseExcludingServiceCostComponent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_NetPeriodicDefinedBenefitsExpenseReversalOfExpenseExcludingServiceCostComponent_b91848b5-c3bf-4601-b0e7-3954cfcf4885" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost_a9eaa0fd-a9f4-445c-9413-7dad2f909deb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_7774f7a4-0656-41ad-bd72-a4dea3ba4dbc" xlink:to="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost_a9eaa0fd-a9f4-445c-9413-7dad2f909deb" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_3472d4b5-f206-4054-bafd-d0c27560f4f9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_ebf8e9a3-83dc-44bb-8af3-6b4ed5003534" xlink:to="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_3472d4b5-f206-4054-bafd-d0c27560f4f9" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeAxis_bb47666c-9b18-447e-82e7-54f89ae01562" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetirementPlanTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_3472d4b5-f206-4054-bafd-d0c27560f4f9" xlink:to="loc_us-gaap_RetirementPlanTypeAxis_bb47666c-9b18-447e-82e7-54f89ae01562" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeDomain_bb47666c-9b18-447e-82e7-54f89ae01562_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetirementPlanTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RetirementPlanTypeAxis_bb47666c-9b18-447e-82e7-54f89ae01562" xlink:to="loc_us-gaap_RetirementPlanTypeDomain_bb47666c-9b18-447e-82e7-54f89ae01562_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeDomain_745a32d2-8008-424e-b0e3-ab8d697c758d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RetirementPlanTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RetirementPlanTypeAxis_bb47666c-9b18-447e-82e7-54f89ae01562" xlink:to="loc_us-gaap_RetirementPlanTypeDomain_745a32d2-8008-424e-b0e3-ab8d697c758d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PensionPlansDefinedBenefitMember_a58c4e6f-5f4b-45fa-933e-3d197ae0f9d8" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PensionPlansDefinedBenefitMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RetirementPlanTypeDomain_745a32d2-8008-424e-b0e3-ab8d697c758d" xlink:to="loc_us-gaap_PensionPlansDefinedBenefitMember_a58c4e6f-5f4b-45fa-933e-3d197ae0f9d8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherPostretirementBenefitPlansDefinedBenefitMember_08fe204a-0a52-4317-9c58-c724c5a6549a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OtherPostretirementBenefitPlansDefinedBenefitMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RetirementPlanTypeDomain_745a32d2-8008-424e-b0e3-ab8d697c758d" xlink:to="loc_us-gaap_OtherPostretirementBenefitPlansDefinedBenefitMember_08fe204a-0a52-4317-9c58-c724c5a6549a" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsActivityRelatedtoLongTermDebtDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#DebtandCreditAgreementsActivityRelatedtoLongTermDebtDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsActivityRelatedtoLongTermDebtDetails" xlink:type="extended" id="if8751af1d8864a09bbb47ac607e8ce82_DebtandCreditAgreementsActivityRelatedtoLongTermDebtDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_8cc5132a-8cc5-4655-af2b-27d3ebb736c1" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:href="csx-20220331.xsd#csx_MovementDebtInstrumentsRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_8cc5132a-8cc5-4655-af2b-27d3ebb736c1" xlink:to="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent_7c993311-2619-4808-8d6e-2ce209c93aaf" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:to="loc_us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent_7c993311-2619-4808-8d6e-2ce209c93aaf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligations_f0f3eabd-10c1-41f5-b6ea-70c410a074ee" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligations"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:to="loc_us-gaap_LongTermDebtAndCapitalLeaseObligations_f0f3eabd-10c1-41f5-b6ea-70c410a074ee" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtAndCapitalLeaseObligations_d47de0e5-d30a-449a-a951-ac45ab1fb212" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtAndCapitalLeaseObligations"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:to="loc_us-gaap_DebtAndCapitalLeaseObligations_d47de0e5-d30a-449a-a951-ac45ab1fb212" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RepaymentsOfLongTermDebt_ee835c69-92c9-42c8-96da-05c9cd8dc1d6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RepaymentsOfLongTermDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:to="loc_us-gaap_RepaymentsOfLongTermDebt_ee835c69-92c9-42c8-96da-05c9cd8dc1d6" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongTermDebtIncreaseDecreaseDueToReclassifications_b1d11ba4-cc8f-4de4-b8bc-534d5fbae48b" xlink:href="csx-20220331.xsd#csx_LongTermDebtIncreaseDecreaseDueToReclassifications"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:to="loc_csx_LongTermDebtIncreaseDecreaseDueToReclassifications_b1d11ba4-cc8f-4de4-b8bc-534d5fbae48b" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AmortizationOfDebtDiscountPremium_0b16eada-f66f-4161-8c59-947014dfff1a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AmortizationOfDebtDiscountPremium"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_1cc1c90b-7347-487e-8eb2-7136f009d896" xlink:to="loc_us-gaap_AmortizationOfDebtDiscountPremium_0b16eada-f66f-4161-8c59-947014dfff1a" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent_44d88db7-d4b7-4310-883a-6a0ad07e09e7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligations_3a213759-1d64-4352-a96a-af4d06101d5d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligations"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtAndCapitalLeaseObligations_1ae5b6b4-b2cc-4b6d-bc2f-d0bb40cea8cc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtAndCapitalLeaseObligations"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_eb8ed123-99fe-4c52-85e9-793acf6c603d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_8cc5132a-8cc5-4655-af2b-27d3ebb736c1" xlink:to="loc_us-gaap_DebtInstrumentTable_eb8ed123-99fe-4c52-85e9-793acf6c603d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationAxis_151a5810-6096-4a73-a4e7-882398203640" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BalanceSheetLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_eb8ed123-99fe-4c52-85e9-793acf6c603d" xlink:to="loc_us-gaap_BalanceSheetLocationAxis_151a5810-6096-4a73-a4e7-882398203640" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_151a5810-6096-4a73-a4e7-882398203640_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_151a5810-6096-4a73-a4e7-882398203640" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_151a5810-6096-4a73-a4e7-882398203640_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BalanceSheetLocationDomain_4dc00ca4-d4b4-4b39-ba32-fcf35ffbacd6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BalanceSheetLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BalanceSheetLocationAxis_151a5810-6096-4a73-a4e7-882398203640" xlink:to="loc_us-gaap_BalanceSheetLocationDomain_4dc00ca4-d4b4-4b39-ba32-fcf35ffbacd6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CurrentLiabilitiesMember_20e19ffe-6071-4f4b-bd61-0c6269d4029b" xlink:href="csx-20220331.xsd#csx_CurrentLiabilitiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_4dc00ca4-d4b4-4b39-ba32-fcf35ffbacd6" xlink:to="loc_csx_CurrentLiabilitiesMember_20e19ffe-6071-4f4b-bd61-0c6269d4029b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongTermLiabilitiesMember_7c944eee-43fd-4faf-851f-83be56ebe4bc" xlink:href="csx-20220331.xsd#csx_LongTermLiabilitiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BalanceSheetLocationDomain_4dc00ca4-d4b4-4b39-ba32-fcf35ffbacd6" xlink:to="loc_csx_LongTermLiabilitiesMember_7c944eee-43fd-4faf-851f-83be56ebe4bc" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#DebtandCreditAgreementsNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsNarrativeDetails" xlink:type="extended" id="iccf0ed0644fa4cd69f2b60db022a0a1d_DebtandCreditAgreementsNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LineOfCreditFacilityLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DerivativeNumberOfFairValueHedges_ac05caee-c7df-48a3-beea-21557f568aae" xlink:href="csx-20220331.xsd#csx_DerivativeNumberOfFairValueHedges"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_csx_DerivativeNumberOfFairValueHedges_ac05caee-c7df-48a3-beea-21557f568aae" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeTermOfContract_5030fc0a-970d-4bb3-8b32-8dbde25284bc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeTermOfContract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_DerivativeTermOfContract_5030fc0a-970d-4bb3-8b32-8dbde25284bc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_9b40a722-c843-4cea-9cf6-d7064bc1a953" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_9b40a722-c843-4cea-9cf6-d7064bc1a953" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue_48cdacbb-c8bd-452b-acfd-31206f2b860d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue_48cdacbb-c8bd-452b-acfd-31206f2b860d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeAssetNotionalAmount_31bbf8a8-8c2c-42dc-ad58-a0851b99e790" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeAssetNotionalAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_DerivativeAssetNotionalAmount_31bbf8a8-8c2c-42dc-ad58-a0851b99e790" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentInterestRateStatedPercentage_727dba76-88ef-46df-b413-59ec31e5df9f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_DebtInstrumentInterestRateStatedPercentage_727dba76-88ef-46df-b413-59ec31e5df9f" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashFlowHedgeDerivativeInstrumentAssetsAtFairValue_7cde112f-ddd9-4804-90b3-3834c3dd2640" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CashFlowHedgeDerivativeInstrumentAssetsAtFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_CashFlowHedgeDerivativeInstrumentAssetsAtFairValue_7cde112f-ddd9-4804-90b3-3834c3dd2640" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossBeforeReclassificationAfterTax_626de0ee-6cd1-4bd2-9461-c603cd241974" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossBeforeReclassificationAfterTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossCashFlowHedgeGainLossBeforeReclassificationAfterTax_626de0ee-6cd1-4bd2-9461-c603cd241974" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_382927e7-71c8-44e6-b49c-6015b5fa4cdc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_382927e7-71c8-44e6-b49c-6015b5fa4cdc" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCredit_e3bf847e-023e-41a7-9a96-d4d48cbb4495" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LineOfCredit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_LineOfCredit_e3bf847e-023e-41a7-9a96-d4d48cbb4495" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_LineOfCreditFacilityTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_de76e956-d1f4-462e-841b-acbf15755ace" xlink:to="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationAxis_94d3ca0f-a280-4fa2-a402-954c3aa0175d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:to="loc_us-gaap_HedgingDesignationAxis_94d3ca0f-a280-4fa2-a402-954c3aa0175d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_94d3ca0f-a280-4fa2-a402-954c3aa0175d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_HedgingDesignationAxis_94d3ca0f-a280-4fa2-a402-954c3aa0175d" xlink:to="loc_us-gaap_HedgingDesignationDomain_94d3ca0f-a280-4fa2-a402-954c3aa0175d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_199b39e8-f944-4bd8-9c8c-fdbe302d1b8d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_HedgingDesignationAxis_94d3ca0f-a280-4fa2-a402-954c3aa0175d" xlink:to="loc_us-gaap_HedgingDesignationDomain_199b39e8-f944-4bd8-9c8c-fdbe302d1b8d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DesignatedAsHedgingInstrumentMember_fcb762a5-5e55-418a-86dd-0e8b574d9a5c" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DesignatedAsHedgingInstrumentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingDesignationDomain_199b39e8-f944-4bd8-9c8c-fdbe302d1b8d" xlink:to="loc_us-gaap_DesignatedAsHedgingInstrumentMember_fcb762a5-5e55-418a-86dd-0e8b574d9a5c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_cffcb20a-839e-4e9e-9f1d-e7cb84227cff" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_cffcb20a-839e-4e9e-9f1d-e7cb84227cff" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_cffcb20a-839e-4e9e-9f1d-e7cb84227cff_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_cffcb20a-839e-4e9e-9f1d-e7cb84227cff" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_cffcb20a-839e-4e9e-9f1d-e7cb84227cff_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_6b24e173-cdcb-4443-bc1d-3fa09950f44b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_cffcb20a-839e-4e9e-9f1d-e7cb84227cff" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_6b24e173-cdcb-4443-bc1d-3fa09950f44b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedToFloatingInterestRateSwapMember_4381e890-abf1-4d55-a1f7-ca22f96bf7ea" xlink:href="csx-20220331.xsd#csx_FixedToFloatingInterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_6b24e173-cdcb-4443-bc1d-3fa09950f44b" xlink:to="loc_csx_FixedToFloatingInterestRateSwapMember_4381e890-abf1-4d55-a1f7-ca22f96bf7ea" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ForwardStartingInterestRateSwapMember_8e184b73-cee6-48a1-9172-acab6d680388" xlink:href="csx-20220331.xsd#csx_ForwardStartingInterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_6b24e173-cdcb-4443-bc1d-3fa09950f44b" xlink:to="loc_csx_ForwardStartingInterestRateSwapMember_8e184b73-cee6-48a1-9172-acab6d680388" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_395dd5e5-7111-4f74-bb9f-5d7e92754489" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:to="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_395dd5e5-7111-4f74-bb9f-5d7e92754489" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_395dd5e5-7111-4f74-bb9f-5d7e92754489_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_395dd5e5-7111-4f74-bb9f-5d7e92754489" xlink:to="loc_us-gaap_HedgingRelationshipDomain_395dd5e5-7111-4f74-bb9f-5d7e92754489_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_61e372b5-10b1-45e1-be6c-f2659ee7ba63" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_395dd5e5-7111-4f74-bb9f-5d7e92754489" xlink:to="loc_us-gaap_HedgingRelationshipDomain_61e372b5-10b1-45e1-be6c-f2659ee7ba63" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueHedgingMember_9f70e529-94bb-46d9-a347-5cc38dcdb914" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_61e372b5-10b1-45e1-be6c-f2659ee7ba63" xlink:to="loc_us-gaap_FairValueHedgingMember_9f70e529-94bb-46d9-a347-5cc38dcdb914" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashFlowHedgingMember_1e5044f5-d487-48b5-b8c9-86d1aebb62a7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CashFlowHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_61e372b5-10b1-45e1-be6c-f2659ee7ba63" xlink:to="loc_us-gaap_CashFlowHedgingMember_1e5044f5-d487-48b5-b8c9-86d1aebb62a7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_8dce1354-69ff-429f-8a71-2997f5a4b511" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:to="loc_us-gaap_DebtInstrumentAxis_8dce1354-69ff-429f-8a71-2997f5a4b511" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_8dce1354-69ff-429f-8a71-2997f5a4b511_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_8dce1354-69ff-429f-8a71-2997f5a4b511" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_8dce1354-69ff-429f-8a71-2997f5a4b511_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_b5dedae9-fe61-4b49-97c2-6e15e6e4318f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_8dce1354-69ff-429f-8a71-2997f5a4b511" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_b5dedae9-fe61-4b49-97c2-6e15e6e4318f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedRateNotesDueBetween2036And2040Member_e74415fc-0437-486a-a79d-75fe03d325fd" xlink:href="csx-20220331.xsd#csx_FixedRateNotesDueBetween2036And2040Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_b5dedae9-fe61-4b49-97c2-6e15e6e4318f" xlink:to="loc_csx_FixedRateNotesDueBetween2036And2040Member_e74415fc-0437-486a-a79d-75fe03d325fd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_A325NotesDue2027Member_bf73e74e-8591-4fd1-803e-714ca6fe20f4" xlink:href="csx-20220331.xsd#csx_A325NotesDue2027Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_b5dedae9-fe61-4b49-97c2-6e15e6e4318f" xlink:to="loc_csx_A325NotesDue2027Member_bf73e74e-8591-4fd1-803e-714ca6fe20f4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_UnsecuredRevolvingCreditFacilityDue2024Member_15ce4079-65c6-4762-8db4-73beb4601134" xlink:href="csx-20220331.xsd#csx_UnsecuredRevolvingCreditFacilityDue2024Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_b5dedae9-fe61-4b49-97c2-6e15e6e4318f" xlink:to="loc_csx_UnsecuredRevolvingCreditFacilityDue2024Member_15ce4079-65c6-4762-8db4-73beb4601134" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityAxis_4c7c8bd1-ab0f-4864-b248-a84a1c14eaec" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CreditFacilityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_f6a2e1dd-a7f6-4f48-86dc-dd6ab52d924d" xlink:to="loc_us-gaap_CreditFacilityAxis_4c7c8bd1-ab0f-4864-b248-a84a1c14eaec" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_4c7c8bd1-ab0f-4864-b248-a84a1c14eaec_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CreditFacilityAxis_4c7c8bd1-ab0f-4864-b248-a84a1c14eaec" xlink:to="loc_us-gaap_CreditFacilityDomain_4c7c8bd1-ab0f-4864-b248-a84a1c14eaec_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_12cf9292-a2c9-4eb4-b827-5a243143d277" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CreditFacilityAxis_4c7c8bd1-ab0f-4864-b248-a84a1c14eaec" xlink:to="loc_us-gaap_CreditFacilityDomain_12cf9292-a2c9-4eb4-b827-5a243143d277" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevolvingCreditFacilityMember_20f2ece8-f1da-46f7-b83d-4bea75345738" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RevolvingCreditFacilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_12cf9292-a2c9-4eb4-b827-5a243143d277" xlink:to="loc_us-gaap_RevolvingCreditFacilityMember_20f2ece8-f1da-46f7-b83d-4bea75345738" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_2a9cd418-259f-407f-9539-3520dc27e02f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_12cf9292-a2c9-4eb4-b827-5a243143d277" xlink:to="loc_us-gaap_CommercialPaperMember_2a9cd418-259f-407f-9539-3520dc27e02f" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails" xlink:type="extended" id="iae10f28529fe4f7e8314aa802ee0b6cb_DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLineItems_9704fc48-025f-4e05-8110-bd1dbe6ec9bc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_ec83b896-e548-4570-81a1-7c344fc7270b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_9704fc48-025f-4e05-8110-bd1dbe6ec9bc" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_ec83b896-e548-4570-81a1-7c344fc7270b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgedLiabilityFairValueHedgeCumulativeIncreaseDecrease_11f7a381-1a82-4639-ab75-147c94f453fe" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgedLiabilityFairValueHedgeCumulativeIncreaseDecrease"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_9704fc48-025f-4e05-8110-bd1dbe6ec9bc" xlink:to="loc_us-gaap_HedgedLiabilityFairValueHedgeCumulativeIncreaseDecrease_11f7a381-1a82-4639-ab75-147c94f453fe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgedLiabilityFairValueHedge_af1ee884-16bc-4355-878f-6ff945962486" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgedLiabilityFairValueHedge"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_9704fc48-025f-4e05-8110-bd1dbe6ec9bc" xlink:to="loc_us-gaap_HedgedLiabilityFairValueHedge_af1ee884-16bc-4355-878f-6ff945962486" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeTable_eb30eb36-2bd7-48ae-b6bc-39f882cd750e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DerivativeLineItems_9704fc48-025f-4e05-8110-bd1dbe6ec9bc" xlink:to="loc_us-gaap_DerivativeTable_eb30eb36-2bd7-48ae-b6bc-39f882cd750e" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_60944113-b928-4f40-8f1f-10e50622fe0d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DerivativeTable_eb30eb36-2bd7-48ae-b6bc-39f882cd750e" xlink:to="loc_us-gaap_DebtInstrumentAxis_60944113-b928-4f40-8f1f-10e50622fe0d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_60944113-b928-4f40-8f1f-10e50622fe0d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_60944113-b928-4f40-8f1f-10e50622fe0d" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_60944113-b928-4f40-8f1f-10e50622fe0d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_4ae9e4ac-6364-45e4-93c9-d3bee824af79" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_60944113-b928-4f40-8f1f-10e50622fe0d" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_4ae9e4ac-6364-45e4-93c9-d3bee824af79" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedRateNotesDueBetween2036And2040Member_2abfede1-8fd4-4c7a-bc63-9b3586475915" xlink:href="csx-20220331.xsd#csx_FixedRateNotesDueBetween2036And2040Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_4ae9e4ac-6364-45e4-93c9-d3bee824af79" xlink:to="loc_csx_FixedRateNotesDueBetween2036And2040Member_2abfede1-8fd4-4c7a-bc63-9b3586475915" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_8f9f2e5c-0381-4b05-9d5e-2f4f99e9c7da" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DerivativeTable_eb30eb36-2bd7-48ae-b6bc-39f882cd750e" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_8f9f2e5c-0381-4b05-9d5e-2f4f99e9c7da" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_8f9f2e5c-0381-4b05-9d5e-2f4f99e9c7da_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_8f9f2e5c-0381-4b05-9d5e-2f4f99e9c7da" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_8f9f2e5c-0381-4b05-9d5e-2f4f99e9c7da_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_b92987dc-e86a-4013-aa10-da5efea36296" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_8f9f2e5c-0381-4b05-9d5e-2f4f99e9c7da" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_b92987dc-e86a-4013-aa10-da5efea36296" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedToFloatingInterestRateSwapMember_3e803ba1-b2aa-4f18-99a2-3a82acdde3de" xlink:href="csx-20220331.xsd#csx_FixedToFloatingInterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_b92987dc-e86a-4013-aa10-da5efea36296" xlink:to="loc_csx_FixedToFloatingInterestRateSwapMember_3e803ba1-b2aa-4f18-99a2-3a82acdde3de" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_e792d375-0c09-4fe3-986f-22b981b49e40" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DerivativeTable_eb30eb36-2bd7-48ae-b6bc-39f882cd750e" xlink:to="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_e792d375-0c09-4fe3-986f-22b981b49e40" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_e792d375-0c09-4fe3-986f-22b981b49e40_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_e792d375-0c09-4fe3-986f-22b981b49e40" xlink:to="loc_us-gaap_HedgingRelationshipDomain_e792d375-0c09-4fe3-986f-22b981b49e40_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_bc714c57-7672-4f04-9623-3a9b735cdf7a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_e792d375-0c09-4fe3-986f-22b981b49e40" xlink:to="loc_us-gaap_HedgingRelationshipDomain_bc714c57-7672-4f04-9623-3a9b735cdf7a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueHedgingMember_c80de72d-603f-4ace-8a6a-037b7f38d299" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_bc714c57-7672-4f04-9623-3a9b735cdf7a" xlink:to="loc_us-gaap_FairValueHedgingMember_c80de72d-603f-4ace-8a6a-037b7f38d299" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationAxis_18143c7c-696b-44ca-bfa6-0daf9dd533eb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DerivativeTable_eb30eb36-2bd7-48ae-b6bc-39f882cd750e" xlink:to="loc_us-gaap_HedgingDesignationAxis_18143c7c-696b-44ca-bfa6-0daf9dd533eb" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_18143c7c-696b-44ca-bfa6-0daf9dd533eb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_HedgingDesignationAxis_18143c7c-696b-44ca-bfa6-0daf9dd533eb" xlink:to="loc_us-gaap_HedgingDesignationDomain_18143c7c-696b-44ca-bfa6-0daf9dd533eb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_e4f75ee2-4246-4438-946a-d58ad599a0f5" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_HedgingDesignationAxis_18143c7c-696b-44ca-bfa6-0daf9dd533eb" xlink:to="loc_us-gaap_HedgingDesignationDomain_e4f75ee2-4246-4438-946a-d58ad599a0f5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DesignatedAsHedgingInstrumentMember_03fc69bd-d049-4481-b7d1-0d9ba9e88f43" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DesignatedAsHedgingInstrumentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingDesignationDomain_e4f75ee2-4246-4438-946a-d58ad599a0f5" xlink:to="loc_us-gaap_DesignatedAsHedgingInstrumentMember_03fc69bd-d049-4481-b7d1-0d9ba9e88f43" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/RevenuesRevenuesDisaggregatedbyLinesofBusinessDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#RevenuesRevenuesDisaggregatedbyLinesofBusinessDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/RevenuesRevenuesDisaggregatedbyLinesofBusinessDetails" xlink:type="extended" id="i7af81eec7a3e4662b65cd5737a9b48d3_RevenuesRevenuesDisaggregatedbyLinesofBusinessDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_b35b22f1-46df-4557-bd8e-88dd6cb47832" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_f55e2251-fcb5-4d15-b372-4dc76ffbb36e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_b35b22f1-46df-4557-bd8e-88dd6cb47832" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_f55e2251-fcb5-4d15-b372-4dc76ffbb36e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_e6b87d81-b3eb-4c61-a4c4-87471a486a38" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_b35b22f1-46df-4557-bd8e-88dd6cb47832" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_e6b87d81-b3eb-4c61-a4c4-87471a486a38" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_527d3717-b87a-45d2-bdac-cb6715c333ec" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_e6b87d81-b3eb-4c61-a4c4-87471a486a38" xlink:to="loc_srt_ProductOrServiceAxis_527d3717-b87a-45d2-bdac-cb6715c333ec" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_527d3717-b87a-45d2-bdac-cb6715c333ec_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_527d3717-b87a-45d2-bdac-cb6715c333ec" xlink:to="loc_srt_ProductsAndServicesDomain_527d3717-b87a-45d2-bdac-cb6715c333ec_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_527d3717-b87a-45d2-bdac-cb6715c333ec" xlink:to="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:href="csx-20220331.xsd#csx_TotalMerchandiseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:to="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ChemicalsMember_aaa1fdc0-9068-4e38-8b8c-168a0eb72c5c" xlink:href="csx-20220331.xsd#csx_ChemicalsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_ChemicalsMember_aaa1fdc0-9068-4e38-8b8c-168a0eb72c5c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_AgriculturalandFoodProductsMember_0e2b9f02-fdec-4188-a57b-309aaf534435" xlink:href="csx-20220331.xsd#csx_AgriculturalandFoodProductsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_AgriculturalandFoodProductsMember_0e2b9f02-fdec-4188-a57b-309aaf534435" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ForestProductsMember_7d689272-1bfb-43cc-aa68-861c3d3b8505" xlink:href="csx-20220331.xsd#csx_ForestProductsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_ForestProductsMember_7d689272-1bfb-43cc-aa68-861c3d3b8505" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_AutomotiveMember_792bfcf2-6930-4bf8-9bbf-56ada33d7f33" xlink:href="csx-20220331.xsd#csx_AutomotiveMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_AutomotiveMember_792bfcf2-6930-4bf8-9bbf-56ada33d7f33" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_MetalsandEquipmentMember_62a57f40-d9c1-4652-9bdf-8bd93efa4b7e" xlink:href="csx-20220331.xsd#csx_MetalsandEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_MetalsandEquipmentMember_62a57f40-d9c1-4652-9bdf-8bd93efa4b7e" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_MineralsMember_68d1118d-e355-4ac4-9b7e-5c8211386cc7" xlink:href="csx-20220331.xsd#csx_MineralsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_MineralsMember_68d1118d-e355-4ac4-9b7e-5c8211386cc7" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FertilizersMember_c6741b0e-0050-4a88-adde-5ed857a9a3b4" xlink:href="csx-20220331.xsd#csx_FertilizersMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_34bc06ab-c184-4eb9-a022-a6aaf29352cb" xlink:to="loc_csx_FertilizersMember_c6741b0e-0050-4a88-adde-5ed857a9a3b4" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_IntermodalMember_78c80745-26b5-412e-a068-148190462ef7" xlink:href="csx-20220331.xsd#csx_IntermodalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:to="loc_csx_IntermodalMember_78c80745-26b5-412e-a068-148190462ef7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CoalServicesMember_da113b3c-2523-4ca4-b506-b2e85b77e9ea" xlink:href="csx-20220331.xsd#csx_CoalServicesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:to="loc_csx_CoalServicesMember_da113b3c-2523-4ca4-b506-b2e85b77e9ea" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TruckingMember_c4815b57-78f3-43b5-b8dd-308d485f03f9" xlink:href="csx-20220331.xsd#csx_TruckingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:to="loc_csx_TruckingMember_c4815b57-78f3-43b5-b8dd-308d485f03f9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OtherServicesMember_6e6bdc9b-fe9a-4a5c-8bfa-9a8f6213908e" xlink:href="csx-20220331.xsd#csx_OtherServicesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_2d912b25-0c95-4671-887f-2300f54dbe74" xlink:to="loc_csx_OtherServicesMember_6e6bdc9b-fe9a-4a5c-8bfa-9a8f6213908e" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/RevenuesNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#RevenuesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/RevenuesNarrativeDetails" xlink:type="extended" id="iaee6d7274d0c473e97e8b75f46b74b6e_RevenuesNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_a69b76b6-463a-4f3d-9afa-4731cf40bf90" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_RevenuePerformanceObligationAverageTransitTime_543f0944-e49f-47e7-8c46-c2698d7c843c" xlink:href="csx-20220331.xsd#csx_RevenuePerformanceObligationAverageTransitTime"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_a69b76b6-463a-4f3d-9afa-4731cf40bf90" xlink:to="loc_csx_RevenuePerformanceObligationAverageTransitTime_543f0944-e49f-47e7-8c46-c2698d7c843c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ContractWithCustomerTimingOfSatisfactionOfPerformanceObligationPaymentPeriod_0f538ce3-b039-435f-aef7-26ccda2437bd" xlink:href="csx-20220331.xsd#csx_ContractWithCustomerTimingOfSatisfactionOfPerformanceObligationPaymentPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_a69b76b6-463a-4f3d-9afa-4731cf40bf90" xlink:to="loc_csx_ContractWithCustomerTimingOfSatisfactionOfPerformanceObligationPaymentPeriod_0f538ce3-b039-435f-aef7-26ccda2437bd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenuePerformanceObligationDescriptionOfTiming_951be335-d6e6-4132-a761-cb71c3dadbd3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RevenuePerformanceObligationDescriptionOfTiming"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_a69b76b6-463a-4f3d-9afa-4731cf40bf90" xlink:to="loc_us-gaap_RevenuePerformanceObligationDescriptionOfTiming_951be335-d6e6-4132-a761-cb71c3dadbd3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsReceivableGross_b3109146-eb5c-4cfa-8992-3aaaf5654886" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccountsReceivableGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_a69b76b6-463a-4f3d-9afa-4731cf40bf90" xlink:to="loc_us-gaap_AccountsReceivableGross_b3109146-eb5c-4cfa-8992-3aaaf5654886" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_6ba98b0d-00d9-44f4-8d78-7b83d081e390" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_a69b76b6-463a-4f3d-9afa-4731cf40bf90" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_6ba98b0d-00d9-44f4-8d78-7b83d081e390" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_aba0a70e-be0c-4d18-a393-2e033a3ae1a6" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_6ba98b0d-00d9-44f4-8d78-7b83d081e390" xlink:to="loc_srt_RangeAxis_aba0a70e-be0c-4d18-a393-2e033a3ae1a6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_aba0a70e-be0c-4d18-a393-2e033a3ae1a6_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_aba0a70e-be0c-4d18-a393-2e033a3ae1a6" xlink:to="loc_srt_RangeMember_aba0a70e-be0c-4d18-a393-2e033a3ae1a6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_1c58c2cb-8d58-441f-b77f-b455ada87c96" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_aba0a70e-be0c-4d18-a393-2e033a3ae1a6" xlink:to="loc_srt_RangeMember_1c58c2cb-8d58-441f-b77f-b455ada87c96" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_002b4e24-e0ce-450c-8e6c-3a1e6f502145" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_1c58c2cb-8d58-441f-b77f-b455ada87c96" xlink:to="loc_srt_MinimumMember_002b4e24-e0ce-450c-8e6c-3a1e6f502145" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_5d550a0f-a53c-45cc-b87c-e1d2e9fdf91b" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_1c58c2cb-8d58-441f-b77f-b455ada87c96" xlink:to="loc_srt_MaximumMember_5d550a0f-a53c-45cc-b87c-e1d2e9fdf91b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_0fe85793-1e95-4e21-9e66-0c7f6ab73336" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_6ba98b0d-00d9-44f4-8d78-7b83d081e390" xlink:to="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_0fe85793-1e95-4e21-9e66-0c7f6ab73336" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReceivableTypeDomain_0fe85793-1e95-4e21-9e66-0c7f6ab73336_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReceivableTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_0fe85793-1e95-4e21-9e66-0c7f6ab73336" xlink:to="loc_us-gaap_ReceivableTypeDomain_0fe85793-1e95-4e21-9e66-0c7f6ab73336_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReceivableTypeDomain_073bca03-e966-4cc7-8ec3-d266f545f09d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReceivableTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_0fe85793-1e95-4e21-9e66-0c7f6ab73336" xlink:to="loc_us-gaap_ReceivableTypeDomain_073bca03-e966-4cc7-8ec3-d266f545f09d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NonFreightReceivablesMember_4373b227-a798-4c13-af23-d41f95847d90" xlink:href="csx-20220331.xsd#csx_NonFreightReceivablesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReceivableTypeDomain_073bca03-e966-4cc7-8ec3-d266f545f09d" xlink:to="loc_csx_NonFreightReceivablesMember_4373b227-a798-4c13-af23-d41f95847d90" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_923e5f8c-c0f9-4256-a4cf-60aae4babef9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_6ba98b0d-00d9-44f4-8d78-7b83d081e390" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_923e5f8c-c0f9-4256-a4cf-60aae4babef9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_923e5f8c-c0f9-4256-a4cf-60aae4babef9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_923e5f8c-c0f9-4256-a4cf-60aae4babef9" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_923e5f8c-c0f9-4256-a4cf-60aae4babef9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_ed509bfc-b29b-4ba6-90a2-2165caf554df" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_923e5f8c-c0f9-4256-a4cf-60aae4babef9" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_ed509bfc-b29b-4ba6-90a2-2165caf554df" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LandEasementMember_081f2125-baaa-4347-a0a8-62e8d30bf213" xlink:href="csx-20220331.xsd#csx_LandEasementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_ed509bfc-b29b-4ba6-90a2-2165caf554df" xlink:to="loc_csx_LandEasementMember_081f2125-baaa-4347-a0a8-62e8d30bf213" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/RevenuesAccountsReceivableNetDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#RevenuesAccountsReceivableNetDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/RevenuesAccountsReceivableNetDetails" xlink:type="extended" id="iec7cf9b4c9c84a77bc6f52ceee799a04_RevenuesAccountsReceivableNetDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_05d0cd84-5762-4691-b4f8-d620fb809588" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccountsNotesAndLoansReceivableLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsReceivableGross_a924a8b5-9522-44ef-a7ac-2189715b485d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccountsReceivableGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_05d0cd84-5762-4691-b4f8-d620fb809588" xlink:to="loc_us-gaap_AccountsReceivableGross_a924a8b5-9522-44ef-a7ac-2189715b485d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllowanceForDoubtfulAccountsReceivable_dc89f5e5-aa61-4206-8ac4-e49542a903cd" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AllowanceForDoubtfulAccountsReceivable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_05d0cd84-5762-4691-b4f8-d620fb809588" xlink:to="loc_us-gaap_AllowanceForDoubtfulAccountsReceivable_dc89f5e5-aa61-4206-8ac4-e49542a903cd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsReceivableNet_bf62ebfa-f641-4b37-a903-f3c73e2cf2f7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccountsReceivableNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_05d0cd84-5762-4691-b4f8-d620fb809588" xlink:to="loc_us-gaap_AccountsReceivableNet_bf62ebfa-f641-4b37-a903-f3c73e2cf2f7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable_9f112d7d-eb05-46a4-88d7-fc9bbf9829f9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_05d0cd84-5762-4691-b4f8-d620fb809588" xlink:to="loc_us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable_9f112d7d-eb05-46a4-88d7-fc9bbf9829f9" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_c834699f-3277-42c7-b917-f625b5f4d057" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable_9f112d7d-eb05-46a4-88d7-fc9bbf9829f9" xlink:to="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_c834699f-3277-42c7-b917-f625b5f4d057" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReceivableTypeDomain_c834699f-3277-42c7-b917-f625b5f4d057_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReceivableTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_c834699f-3277-42c7-b917-f625b5f4d057" xlink:to="loc_us-gaap_ReceivableTypeDomain_c834699f-3277-42c7-b917-f625b5f4d057_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReceivableTypeDomain_c9dd26a4-859f-411a-8860-d1580629038e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReceivableTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_c834699f-3277-42c7-b917-f625b5f4d057" xlink:to="loc_us-gaap_ReceivableTypeDomain_c9dd26a4-859f-411a-8860-d1580629038e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FreightReceivablesMember_01f7a988-e52e-4d21-a2e6-ab6c8fcaf208" xlink:href="csx-20220331.xsd#csx_FreightReceivablesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReceivableTypeDomain_c9dd26a4-859f-411a-8860-d1580629038e" xlink:to="loc_csx_FreightReceivablesMember_01f7a988-e52e-4d21-a2e6-ab6c8fcaf208" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NonFreightReceivablesMember_1fb9593d-70ec-4c3c-ac31-0d972d33d067" xlink:href="csx-20220331.xsd#csx_NonFreightReceivablesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReceivableTypeDomain_c9dd26a4-859f-411a-8860-d1580629038e" xlink:to="loc_csx_NonFreightReceivablesMember_1fb9593d-70ec-4c3c-ac31-0d972d33d067" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsFairValueofInvestmentAssetsDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#FairValueMeasurementsFairValueofInvestmentAssetsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsFairValueofInvestmentAssetsDetails" xlink:type="extended" id="i6eeb6a0efd264ae7b47715eceac99ff0_FairValueMeasurementsFairValueofInvestmentAssetsDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_c7e12da8-6de9-41d2-bdf0-5f64dd3b42eb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_7db02396-d8bf-4fdc-a00a-4688c37b8975" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_c7e12da8-6de9-41d2-bdf0-5f64dd3b42eb" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_7db02396-d8bf-4fdc-a00a-4688c37b8975" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_6003f23b-7f71-4c63-97d5-feea929c0732" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_c7e12da8-6de9-41d2-bdf0-5f64dd3b42eb" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_6003f23b-7f71-4c63-97d5-feea929c0732" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_7418016b-a73c-43ef-9c8d-c1cea5eebfe1" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_c7e12da8-6de9-41d2-bdf0-5f64dd3b42eb" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_7418016b-a73c-43ef-9c8d-c1cea5eebfe1" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_cc447109-07fc-45c2-b8f6-814a31cc0918" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_7418016b-a73c-43ef-9c8d-c1cea5eebfe1" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_cc447109-07fc-45c2-b8f6-814a31cc0918" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_cc447109-07fc-45c2-b8f6-814a31cc0918_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_cc447109-07fc-45c2-b8f6-814a31cc0918" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_cc447109-07fc-45c2-b8f6-814a31cc0918_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b7740e9f-f86c-452a-96fb-51307a8c12db" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_cc447109-07fc-45c2-b8f6-814a31cc0918" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b7740e9f-f86c-452a-96fb-51307a8c12db" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_c393dd25-140a-4e63-9d9c-1358bebedba4" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b7740e9f-f86c-452a-96fb-51307a8c12db" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_c393dd25-140a-4e63-9d9c-1358bebedba4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_d5ba2eb0-6d48-4257-b295-71bf2b845fc1" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b7740e9f-f86c-452a-96fb-51307a8c12db" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_d5ba2eb0-6d48-4257-b295-71bf2b845fc1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FairValueInputsLevel1AndLevel2Member_2ef84503-82a0-439c-b65a-5cb355c52419" xlink:href="csx-20220331.xsd#csx_FairValueInputsLevel1AndLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_b7740e9f-f86c-452a-96fb-51307a8c12db" xlink:to="loc_csx_FairValueInputsLevel1AndLevel2Member_2ef84503-82a0-439c-b65a-5cb355c52419" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementBasisAxis_c90bf89d-2d07-468b-a305-bbff7bcb9218" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByMeasurementBasisAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_7418016b-a73c-43ef-9c8d-c1cea5eebfe1" xlink:to="loc_us-gaap_FairValueByMeasurementBasisAxis_c90bf89d-2d07-468b-a305-bbff7bcb9218" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_c90bf89d-2d07-468b-a305-bbff7bcb9218_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_c90bf89d-2d07-468b-a305-bbff7bcb9218" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_c90bf89d-2d07-468b-a305-bbff7bcb9218_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureItemAmountsDomain_783c37ff-c567-448a-b622-0645a9c08d0f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueDisclosureItemAmountsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_c90bf89d-2d07-468b-a305-bbff7bcb9218" xlink:to="loc_us-gaap_FairValueDisclosureItemAmountsDomain_783c37ff-c567-448a-b622-0645a9c08d0f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_a33d4988-672a-4467-968b-967b400d07b6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureItemAmountsDomain_783c37ff-c567-448a-b622-0645a9c08d0f" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_a33d4988-672a-4467-968b-967b400d07b6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_c8286e9d-def3-4579-8c5e-7cd141acb8f7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EstimateOfFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_a33d4988-672a-4467-968b-967b400d07b6" xlink:to="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_c8286e9d-def3-4579-8c5e-7cd141acb8f7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_f7e5cb34-302e-4b46-b462-76e1a2dd1529" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CarryingReportedAmountFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_a33d4988-672a-4467-968b-967b400d07b6" xlink:to="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_f7e5cb34-302e-4b46-b462-76e1a2dd1529" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentTypeAxis_8277a542-aeb3-4915-baac-2daf1e45e978" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_InvestmentTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_7418016b-a73c-43ef-9c8d-c1cea5eebfe1" xlink:to="loc_us-gaap_InvestmentTypeAxis_8277a542-aeb3-4915-baac-2daf1e45e978" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentTypeCategorizationMember_8277a542-aeb3-4915-baac-2daf1e45e978_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_InvestmentTypeCategorizationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_InvestmentTypeAxis_8277a542-aeb3-4915-baac-2daf1e45e978" xlink:to="loc_us-gaap_InvestmentTypeCategorizationMember_8277a542-aeb3-4915-baac-2daf1e45e978_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentTypeCategorizationMember_48d508c3-6f86-41f4-8951-c3d275f8ca11" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_InvestmentTypeCategorizationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_InvestmentTypeAxis_8277a542-aeb3-4915-baac-2daf1e45e978" xlink:to="loc_us-gaap_InvestmentTypeCategorizationMember_48d508c3-6f86-41f4-8951-c3d275f8ca11" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FixedIncomeFundsMember_7fd985d1-8348-413e-9848-dea0de410cd4" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FixedIncomeFundsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_InvestmentTypeCategorizationMember_48d508c3-6f86-41f4-8951-c3d275f8ca11" xlink:to="loc_us-gaap_FixedIncomeFundsMember_7fd985d1-8348-413e-9848-dea0de410cd4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CorporateBondSecuritiesMember_2cff7267-c947-4ee2-b323-084a7303aa82" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CorporateBondSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_InvestmentTypeCategorizationMember_48d508c3-6f86-41f4-8951-c3d275f8ca11" xlink:to="loc_us-gaap_CorporateBondSecuritiesMember_2cff7267-c947-4ee2-b323-084a7303aa82" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryAndGovernmentMember_b07845b8-a580-4bd3-8129-df5d974924c5" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_USTreasuryAndGovernmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_InvestmentTypeCategorizationMember_48d508c3-6f86-41f4-8951-c3d275f8ca11" xlink:to="loc_us-gaap_USTreasuryAndGovernmentMember_b07845b8-a580-4bd3-8129-df5d974924c5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_3165df5a-2a42-4356-9881-3cc83ff41828" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_InvestmentTypeCategorizationMember_48d508c3-6f86-41f4-8951-c3d275f8ca11" xlink:to="loc_us-gaap_CommercialPaperMember_3165df5a-2a42-4356-9881-3cc83ff41828" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsInvestmentMaturitiesDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#FairValueMeasurementsInvestmentMaturitiesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsInvestmentMaturitiesDetails" xlink:type="extended" id="idafde940089a4a3f9d9e7b6b98065961_FairValueMeasurementsInvestmentMaturitiesDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesNextRollingTwelveMonthsFairValue_4add0dc8-4fac-490f-9e78-9f786cee695d" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleSecuritiesDebtMaturitiesNextRollingTwelveMonthsFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesNextRollingTwelveMonthsFairValue_4add0dc8-4fac-490f-9e78-9f786cee695d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingYearTwoThroughFiveFairValue_57f0fe90-2366-4975-ace8-a2c257a0112b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingYearTwoThroughFiveFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingYearTwoThroughFiveFairValue_57f0fe90-2366-4975-ace8-a2c257a0112b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingYearSixThroughTenFairValue_1d85566a-c232-48c6-ace2-6065a1ef3a40" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingYearSixThroughTenFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingYearSixThroughTenFairValue_1d85566a-c232-48c6-ace2-6065a1ef3a40" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingAfterYearTenFairValue_dee543ea-3610-425f-acb8-9e5f5bc3130a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingAfterYearTenFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtMaturitiesRollingAfterYearTenFairValue_dee543ea-3610-425f-acb8-9e5f5bc3130a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_a08b4a65-338b-4b1a-bd57-dfdcc0e395b7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_a08b4a65-338b-4b1a-bd57-dfdcc0e395b7" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_2551502c-c752-40bb-a0eb-faf69d4495e8" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_cf0947f3-8977-4b04-b4f1-8e897ee7471a" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_2551502c-c752-40bb-a0eb-faf69d4495e8" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementBasisAxis_398f5054-49a1-4b15-8486-d4d055b6804e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByMeasurementBasisAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_2551502c-c752-40bb-a0eb-faf69d4495e8" xlink:to="loc_us-gaap_FairValueByMeasurementBasisAxis_398f5054-49a1-4b15-8486-d4d055b6804e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_398f5054-49a1-4b15-8486-d4d055b6804e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_398f5054-49a1-4b15-8486-d4d055b6804e" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_398f5054-49a1-4b15-8486-d4d055b6804e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureItemAmountsDomain_782f1d22-0e1a-40da-b753-eedd36cf94da" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueDisclosureItemAmountsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_398f5054-49a1-4b15-8486-d4d055b6804e" xlink:to="loc_us-gaap_FairValueDisclosureItemAmountsDomain_782f1d22-0e1a-40da-b753-eedd36cf94da" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_e50a2447-4f7e-49af-9bde-8c6b20fae2e6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureItemAmountsDomain_782f1d22-0e1a-40da-b753-eedd36cf94da" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_e50a2447-4f7e-49af-9bde-8c6b20fae2e6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_0c18530a-d90c-4591-b163-982802110fcc" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EstimateOfFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_e50a2447-4f7e-49af-9bde-8c6b20fae2e6" xlink:to="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_0c18530a-d90c-4591-b163-982802110fcc" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsFairValueandCarryingValueofLongTermDebtDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#FairValueMeasurementsFairValueandCarryingValueofLongTermDebtDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsFairValueandCarryingValueofLongTermDebtDetails" xlink:type="extended" id="i68451a9ad77046e18e6c84791d33ac71_FairValueMeasurementsFairValueandCarryingValueofLongTermDebtDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_f9b1889b-7247-46a0-b5ed-25fd163db7f8" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFairValue_410fce33-8cea-40ee-835b-4b01cc0235cb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DebtInstrumentFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_f9b1889b-7247-46a0-b5ed-25fd163db7f8" xlink:to="loc_us-gaap_DebtInstrumentFairValue_410fce33-8cea-40ee-835b-4b01cc0235cb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_92098e76-b5dd-4ea5-b2e8-6dbb8523cee7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_f9b1889b-7247-46a0-b5ed-25fd163db7f8" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_92098e76-b5dd-4ea5-b2e8-6dbb8523cee7" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_35313a30-4cba-4906-8b7d-f8167cd78d63" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_92098e76-b5dd-4ea5-b2e8-6dbb8523cee7" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_35313a30-4cba-4906-8b7d-f8167cd78d63" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_35313a30-4cba-4906-8b7d-f8167cd78d63_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_35313a30-4cba-4906-8b7d-f8167cd78d63" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_35313a30-4cba-4906-8b7d-f8167cd78d63_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3eecb686-f366-4267-92db-689703af63ed" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_35313a30-4cba-4906-8b7d-f8167cd78d63" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3eecb686-f366-4267-92db-689703af63ed" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_80fe9ae1-319d-4149-b0dd-06940d6ac707" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_3eecb686-f366-4267-92db-689703af63ed" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_80fe9ae1-319d-4149-b0dd-06940d6ac707" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementBasisAxis_ecbefd99-8383-4fc2-917e-ffa244844025" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueByMeasurementBasisAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_92098e76-b5dd-4ea5-b2e8-6dbb8523cee7" xlink:to="loc_us-gaap_FairValueByMeasurementBasisAxis_ecbefd99-8383-4fc2-917e-ffa244844025" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_ecbefd99-8383-4fc2-917e-ffa244844025_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_ecbefd99-8383-4fc2-917e-ffa244844025" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_ecbefd99-8383-4fc2-917e-ffa244844025_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureItemAmountsDomain_ced3eb3d-e1ce-484f-afb2-b8ef9f7fc7e1" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueDisclosureItemAmountsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_ecbefd99-8383-4fc2-917e-ffa244844025" xlink:to="loc_us-gaap_FairValueDisclosureItemAmountsDomain_ced3eb3d-e1ce-484f-afb2-b8ef9f7fc7e1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_6307eb8e-e8c0-4b89-beea-cecfa6662c31" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureItemAmountsDomain_ced3eb3d-e1ce-484f-afb2-b8ef9f7fc7e1" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_6307eb8e-e8c0-4b89-beea-cecfa6662c31" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_0c8f8a0d-7dd6-4dfd-b148-d988c82985d0" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EstimateOfFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_6307eb8e-e8c0-4b89-beea-cecfa6662c31" xlink:to="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_0c8f8a0d-7dd6-4dfd-b148-d988c82985d0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_1976afde-6716-4e37-aabc-2b1b4ce18cd0" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CarryingReportedAmountFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_6307eb8e-e8c0-4b89-beea-cecfa6662c31" xlink:to="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_1976afde-6716-4e37-aabc-2b1b4ce18cd0" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsInterestRateDerivativesDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#FairValueMeasurementsInterestRateDerivativesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsInterestRateDerivativesDetails" xlink:type="extended" id="id2fd2ebb1ffc4272890e165dc191239b_FairValueMeasurementsInterestRateDerivativesDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_234beea2-48dc-46f5-8401-d1ce642fd266" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLiabilityNotionalAmount_b4a438be-79dd-42e3-a882-3319ea8f0aef" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeLiabilityNotionalAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_234beea2-48dc-46f5-8401-d1ce642fd266" xlink:to="loc_us-gaap_DerivativeLiabilityNotionalAmount_b4a438be-79dd-42e3-a882-3319ea8f0aef" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeAssetNotionalAmount_7d35ac12-6e34-4d21-a4be-a96169f5b39f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeAssetNotionalAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_234beea2-48dc-46f5-8401-d1ce642fd266" xlink:to="loc_us-gaap_DerivativeAssetNotionalAmount_7d35ac12-6e34-4d21-a4be-a96169f5b39f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_4c825eb8-082f-47e8-b185-6969722bf4a6" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_234beea2-48dc-46f5-8401-d1ce642fd266" xlink:to="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_4c825eb8-082f-47e8-b185-6969722bf4a6" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_02a29141-9437-4fbd-a713-1beb0faa8b69" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_4c825eb8-082f-47e8-b185-6969722bf4a6" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_02a29141-9437-4fbd-a713-1beb0faa8b69" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_02a29141-9437-4fbd-a713-1beb0faa8b69_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_02a29141-9437-4fbd-a713-1beb0faa8b69" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_02a29141-9437-4fbd-a713-1beb0faa8b69_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_29a49f14-5493-46b2-872e-11046a14a34a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_02a29141-9437-4fbd-a713-1beb0faa8b69" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_29a49f14-5493-46b2-872e-11046a14a34a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedToFloatingInterestRateSwapMember_9ed06ffe-7532-405a-a8c1-84642e15902f" xlink:href="csx-20220331.xsd#csx_FixedToFloatingInterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_29a49f14-5493-46b2-872e-11046a14a34a" xlink:to="loc_csx_FixedToFloatingInterestRateSwapMember_9ed06ffe-7532-405a-a8c1-84642e15902f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ForwardStartingInterestRateSwapMember_e01f8e85-43d6-4089-8cf7-d5e6fe943bb0" xlink:href="csx-20220331.xsd#csx_ForwardStartingInterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_29a49f14-5493-46b2-872e-11046a14a34a" xlink:to="loc_csx_ForwardStartingInterestRateSwapMember_e01f8e85-43d6-4089-8cf7-d5e6fe943bb0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_f5e41c3b-4f2d-4a0f-a091-f17835e29872" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_4c825eb8-082f-47e8-b185-6969722bf4a6" xlink:to="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_f5e41c3b-4f2d-4a0f-a091-f17835e29872" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_f5e41c3b-4f2d-4a0f-a091-f17835e29872_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_f5e41c3b-4f2d-4a0f-a091-f17835e29872" xlink:to="loc_us-gaap_HedgingRelationshipDomain_f5e41c3b-4f2d-4a0f-a091-f17835e29872_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_28f5c188-6d65-4e97-8b0f-2d5c83fe6104" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_f5e41c3b-4f2d-4a0f-a091-f17835e29872" xlink:to="loc_us-gaap_HedgingRelationshipDomain_28f5c188-6d65-4e97-8b0f-2d5c83fe6104" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueHedgingMember_4022a33d-c279-4023-9e88-a6bcd02201b3" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FairValueHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_28f5c188-6d65-4e97-8b0f-2d5c83fe6104" xlink:to="loc_us-gaap_FairValueHedgingMember_4022a33d-c279-4023-9e88-a6bcd02201b3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashFlowHedgingMember_68286176-169d-4279-a012-d0a09940eacb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CashFlowHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_28f5c188-6d65-4e97-8b0f-2d5c83fe6104" xlink:to="loc_us-gaap_CashFlowHedgingMember_68286176-169d-4279-a012-d0a09940eacb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationAxis_0fdffe83-aebc-47ab-a6ac-f2309c368cc7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_4c825eb8-082f-47e8-b185-6969722bf4a6" xlink:to="loc_us-gaap_HedgingDesignationAxis_0fdffe83-aebc-47ab-a6ac-f2309c368cc7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_0fdffe83-aebc-47ab-a6ac-f2309c368cc7_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_HedgingDesignationAxis_0fdffe83-aebc-47ab-a6ac-f2309c368cc7" xlink:to="loc_us-gaap_HedgingDesignationDomain_0fdffe83-aebc-47ab-a6ac-f2309c368cc7_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_8a520b02-06c5-40e4-a12a-04d36a054618" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_HedgingDesignationAxis_0fdffe83-aebc-47ab-a6ac-f2309c368cc7" xlink:to="loc_us-gaap_HedgingDesignationDomain_8a520b02-06c5-40e4-a12a-04d36a054618" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DesignatedAsHedgingInstrumentMember_a12ca44a-20b1-446a-8124-70f24c0e832a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_DesignatedAsHedgingInstrumentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingDesignationDomain_8a520b02-06c5-40e4-a12a-04d36a054618" xlink:to="loc_us-gaap_DesignatedAsHedgingInstrumentMember_a12ca44a-20b1-446a-8124-70f24c0e832a" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/OtherComprehensiveIncomeLossChangesinAOCIBalancebyComponentDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#OtherComprehensiveIncomeLossChangesinAOCIBalancebyComponentDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/OtherComprehensiveIncomeLossChangesinAOCIBalancebyComponentDetails" xlink:type="extended" id="i69c3c78463ee4c5f9aba692312132e7f_OtherComprehensiveIncomeLossChangesinAOCIBalancebyComponentDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems_d77c8397-7c8f-487d-89bd-b4b08bb0b720" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AOCIAttributableToParentNetOfTaxRollForward_6b5b5631-b833-4aad-a826-37128afcaa4a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AOCIAttributableToParentNetOfTaxRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems_d77c8397-7c8f-487d-89bd-b4b08bb0b720" xlink:to="loc_us-gaap_AOCIAttributableToParentNetOfTaxRollForward_6b5b5631-b833-4aad-a826-37128afcaa4a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_af31e1de-fec0-48b9-9881-564601b19fa5" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AOCIAttributableToParentNetOfTaxRollForward_6b5b5631-b833-4aad-a826-37128afcaa4a" xlink:to="loc_us-gaap_StockholdersEquity_af31e1de-fec0-48b9-9881-564601b19fa5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_7fce7a7e-4dc3-4472-8504-9b5cf79e93cf" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AOCIAttributableToParentNetOfTaxRollForward_6b5b5631-b833-4aad-a826-37128afcaa4a" xlink:to="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_7fce7a7e-4dc3-4472-8504-9b5cf79e93cf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OciBeforeReclassificationsBeforeTaxAttributableToParent_e5750e0f-1d1a-4913-8759-7999c0f48a44" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OciBeforeReclassificationsBeforeTaxAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_7fce7a7e-4dc3-4472-8504-9b5cf79e93cf" xlink:to="loc_us-gaap_OciBeforeReclassificationsBeforeTaxAttributableToParent_e5750e0f-1d1a-4913-8759-7999c0f48a44" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationFromAociCurrentPeriodBeforeTaxAttributableToParent_6fe86032-a574-4691-93d3-5173c250d07f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ReclassificationFromAociCurrentPeriodBeforeTaxAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_7fce7a7e-4dc3-4472-8504-9b5cf79e93cf" xlink:to="loc_us-gaap_ReclassificationFromAociCurrentPeriodBeforeTaxAttributableToParent_6fe86032-a574-4691-93d3-5173c250d07f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossTaxPortionAttributableToParent1_ffb3c40a-d242-4e31-9a47-1fccb1d2af6f" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OtherComprehensiveIncomeLossTaxPortionAttributableToParent1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_7fce7a7e-4dc3-4472-8504-9b5cf79e93cf" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossTaxPortionAttributableToParent1_ffb3c40a-d242-4e31-9a47-1fccb1d2af6f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_5265aa4d-9bf7-49fc-b60d-96af5725e3ea" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_7fce7a7e-4dc3-4472-8504-9b5cf79e93cf" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_5265aa4d-9bf7-49fc-b60d-96af5725e3ea" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_ab0fe7cc-b817-47ce-8cb1-02cff4382d52" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StockholdersEquity"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossTable_309fcf09-480a-40d1-aabd-cbc895700815" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems_d77c8397-7c8f-487d-89bd-b4b08bb0b720" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossTable_309fcf09-480a-40d1-aabd-cbc895700815" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_598c10e4-8f38-4afe-a3f7-3785b94bba77" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossTable_309fcf09-480a-40d1-aabd-cbc895700815" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_598c10e4-8f38-4afe-a3f7-3785b94bba77" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_598c10e4-8f38-4afe-a3f7-3785b94bba77_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_598c10e4-8f38-4afe-a3f7-3785b94bba77" xlink:to="loc_us-gaap_EquityComponentDomain_598c10e4-8f38-4afe-a3f7-3785b94bba77_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_e67c5ce5-a9db-422b-96c8-e64d2a7839b7" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_598c10e4-8f38-4afe-a3f7-3785b94bba77" xlink:to="loc_us-gaap_EquityComponentDomain_e67c5ce5-a9db-422b-96c8-e64d2a7839b7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember_82ba39c6-7ad6-473e-b975-f4f62fe6a8eb" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_e67c5ce5-a9db-422b-96c8-e64d2a7839b7" xlink:to="loc_us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember_82ba39c6-7ad6-473e-b975-f4f62fe6a8eb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember_a071c23f-29e1-4c03-84da-11cbabebf356" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_e67c5ce5-a9db-422b-96c8-e64d2a7839b7" xlink:to="loc_us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember_a071c23f-29e1-4c03-84da-11cbabebf356" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_AccumulatedOtherAdjustmentstoParentMember_1f0825d1-0946-4275-802a-05fa6142bed4" xlink:href="csx-20220331.xsd#csx_AccumulatedOtherAdjustmentstoParentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_e67c5ce5-a9db-422b-96c8-e64d2a7839b7" xlink:to="loc_csx_AccumulatedOtherAdjustmentstoParentMember_1f0825d1-0946-4275-802a-05fa6142bed4" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_9568311e-c201-4729-b961-a3fcf3465a36" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_e67c5ce5-a9db-422b-96c8-e64d2a7839b7" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_9568311e-c201-4729-b961-a3fcf3465a36" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/BusinessCombinationsNarrativeDetails" xlink:type="simple" xlink:href="csx-20220331.xsd#BusinessCombinationsNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/BusinessCombinationsNarrativeDetails" xlink:type="extended" id="i6ec3f630f14f44bf89e20d82aada1d1e_BusinessCombinationsNarrativeDetails">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BusinessAcquisitionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsToAcquireBusinessesGross_8c7c490c-a338-4384-98cb-6d638ec5c5c9" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_PaymentsToAcquireBusinessesGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_us-gaap_PaymentsToAcquireBusinessesGross_8c7c490c-a338-4384-98cb-6d638ec5c5c9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAcquiredFromAcquisition_4e3371ae-e905-439c-82b7-d494e5e57b2a" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CashAcquiredFromAcquisition"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_us-gaap_CashAcquiredFromAcquisition_4e3371ae-e905-439c-82b7-d494e5e57b2a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfTerminalsAndFacilities_290e23ad-8fa8-4441-b2cf-33f03a8392d7" xlink:href="csx-20220331.xsd#csx_NumberOfTerminalsAndFacilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_csx_NumberOfTerminalsAndFacilities_290e23ad-8fa8-4441-b2cf-33f03a8392d7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_Goodwill_075db2ba-d7b0-4ec2-bb0c-07c4f85b20d4" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_Goodwill"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_us-gaap_Goodwill_075db2ba-d7b0-4ec2-bb0c-07c4f85b20d4" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinitelivedIntangibleAssetsAcquired1_6a2813e2-fa40-4a61-923c-982ce8fbc978" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FinitelivedIntangibleAssetsAcquired1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_us-gaap_FinitelivedIntangibleAssetsAcquired1_6a2813e2-fa40-4a61-923c-982ce8fbc978" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AcquiredFiniteLivedIntangibleAssetsWeightedAverageUsefulLife_bfa5b77e-db98-4a2b-a963-5e547c73d3d1" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_AcquiredFiniteLivedIntangibleAssetsWeightedAverageUsefulLife"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_us-gaap_AcquiredFiniteLivedIntangibleAssetsWeightedAverageUsefulLife_bfa5b77e-db98-4a2b-a963-5e547c73d3d1" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_RailRouteNetworkDistance_90d3e72a-c864-445e-beb3-88e5f35b87b6" xlink:href="csx-20220331.xsd#csx_RailRouteNetworkDistance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_csx_RailRouteNetworkDistance_90d3e72a-c864-445e-beb3-88e5f35b87b6" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_RailRouteNetworkDistancePartialInterest_227629ad-b268-4a97-bc6c-b265a4daaa54" xlink:href="csx-20220331.xsd#csx_RailRouteNetworkDistancePartialInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_csx_RailRouteNetworkDistancePartialInterest_227629ad-b268-4a97-bc6c-b265a4daaa54" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_80c65c7c-7001-44ad-9563-53b2a10e080b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_BusinessAcquisitionLineItems_f6a2df92-6cea-4d34-b4d0-d5eb9d6131b4" xlink:to="loc_us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_80c65c7c-7001-44ad-9563-53b2a10e080b" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BusinessAcquisitionAxis_36425e6c-d302-4702-8ac1-237eb1689cdf" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BusinessAcquisitionAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_80c65c7c-7001-44ad-9563-53b2a10e080b" xlink:to="loc_us-gaap_BusinessAcquisitionAxis_36425e6c-d302-4702-8ac1-237eb1689cdf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BusinessAcquisitionAcquireeDomain_36425e6c-d302-4702-8ac1-237eb1689cdf_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BusinessAcquisitionAcquireeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_BusinessAcquisitionAxis_36425e6c-d302-4702-8ac1-237eb1689cdf" xlink:to="loc_us-gaap_BusinessAcquisitionAcquireeDomain_36425e6c-d302-4702-8ac1-237eb1689cdf_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BusinessAcquisitionAcquireeDomain_2cac886c-83de-4f7f-afed-f8f005883f93" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_BusinessAcquisitionAcquireeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_BusinessAcquisitionAxis_36425e6c-d302-4702-8ac1-237eb1689cdf" xlink:to="loc_us-gaap_BusinessAcquisitionAcquireeDomain_2cac886c-83de-4f7f-afed-f8f005883f93" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_QualityCarriersIncMember_9c3b8667-375e-4051-9983-d24ce3543685" xlink:href="csx-20220331.xsd#csx_QualityCarriersIncMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionAcquireeDomain_2cac886c-83de-4f7f-afed-f8f005883f93" xlink:to="loc_csx_QualityCarriersIncMember_9c3b8667-375e-4051-9983-d24ce3543685" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_PanAmSystemsIncMember_c8b23022-5836-4c3f-8fe0-a46b70e7cfa9" xlink:href="csx-20220331.xsd#csx_PanAmSystemsIncMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_BusinessAcquisitionAcquireeDomain_2cac886c-83de-4f7f-afed-f8f005883f93" xlink:to="loc_csx_PanAmSystemsIncMember_c8b23022-5836-4c3f-8fe0-a46b70e7cfa9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_LegalEntityAxis_0e83c15f-c921-4666-aff9-38ecbf9bc4b2" xlink:href="https://xbrl.sec.gov/dei/2021q4/dei-2021q4.xsd#dei_LegalEntityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_80c65c7c-7001-44ad-9563-53b2a10e080b" xlink:to="loc_dei_LegalEntityAxis_0e83c15f-c921-4666-aff9-38ecbf9bc4b2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityDomain_0e83c15f-c921-4666-aff9-38ecbf9bc4b2_default" xlink:href="https://xbrl.sec.gov/dei/2021q4/dei-2021q4.xsd#dei_EntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_dei_LegalEntityAxis_0e83c15f-c921-4666-aff9-38ecbf9bc4b2" xlink:to="loc_dei_EntityDomain_0e83c15f-c921-4666-aff9-38ecbf9bc4b2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityDomain_9354b3e1-f64f-4e68-a0b6-eb257c1bface" xlink:href="https://xbrl.sec.gov/dei/2021q4/dei-2021q4.xsd#dei_EntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_dei_LegalEntityAxis_0e83c15f-c921-4666-aff9-38ecbf9bc4b2" xlink:to="loc_dei_EntityDomain_9354b3e1-f64f-4e68-a0b6-eb257c1bface" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_QualityCarriersIncMember_c93b134c-47e2-4edd-9ea7-f40227a743c6" xlink:href="csx-20220331.xsd#csx_QualityCarriersIncMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityDomain_9354b3e1-f64f-4e68-a0b6-eb257c1bface" xlink:to="loc_csx_QualityCarriersIncMember_c93b134c-47e2-4edd-9ea7-f40227a743c6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_PanAmSystemsIncMember_e00f09cd-6f18-4eed-897a-47bd4e9da5a6" xlink:href="csx-20220331.xsd#csx_PanAmSystemsIncMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityDomain_9354b3e1-f64f-4e68-a0b6-eb257c1bface" xlink:to="loc_csx_PanAmSystemsIncMember_e00f09cd-6f18-4eed-897a-47bd4e9da5a6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_35c1c1b2-dec1-4aaf-9167-a069267295af" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_80c65c7c-7001-44ad-9563-53b2a10e080b" xlink:to="loc_srt_StatementGeographicalAxis_35c1c1b2-dec1-4aaf-9167-a069267295af" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_35c1c1b2-dec1-4aaf-9167-a069267295af_default" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_35c1c1b2-dec1-4aaf-9167-a069267295af" xlink:to="loc_srt_SegmentGeographicalDomain_35c1c1b2-dec1-4aaf-9167-a069267295af_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_454e2d24-e639-4a03-8535-3b0337815c3e" xlink:href="https://xbrl.fasb.org/srt/2021/elts/srt-2021-01-31.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_35c1c1b2-dec1-4aaf-9167-a069267295af" xlink:to="loc_srt_SegmentGeographicalDomain_454e2d24-e639-4a03-8535-3b0337815c3e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_UnitedStatesCanadaAndMexicoMember_227a0645-8529-42b2-924d-c8ee080a6f23" xlink:href="csx-20220331.xsd#csx_UnitedStatesCanadaAndMexicoMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_454e2d24-e639-4a03-8535-3b0337815c3e" xlink:to="loc_csx_UnitedStatesCanadaAndMexicoMember_227a0645-8529-42b2-924d-c8ee080a6f23" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_770c4a73-940b-44c1-bd66-f4902fdb869e" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable_80c65c7c-7001-44ad-9563-53b2a10e080b" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_770c4a73-940b-44c1-bd66-f4902fdb869e" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_770c4a73-940b-44c1-bd66-f4902fdb869e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_770c4a73-940b-44c1-bd66-f4902fdb869e" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_770c4a73-940b-44c1-bd66-f4902fdb869e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_09ee35f4-2df8-476f-a08b-fc2f02ee039c" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis_770c4a73-940b-44c1-bd66-f4902fdb869e" xlink:to="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_09ee35f4-2df8-476f-a08b-fc2f02ee039c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CustomerRelationshipsMember_16fa6f83-8aad-457c-93f0-17d3ed30ce8b" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_CustomerRelationshipsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_09ee35f4-2df8-476f-a08b-fc2f02ee039c" xlink:to="loc_us-gaap_CustomerRelationshipsMember_16fa6f83-8aad-457c-93f0-17d3ed30ce8b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TradeNamesMember_6a5b0f7b-22b7-49e9-8291-9e592b1ff5f1" xlink:href="https://xbrl.fasb.org/us-gaap/2021/elts/us-gaap-2021-01-31.xsd#us-gaap_TradeNamesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_09ee35f4-2df8-476f-a08b-fc2f02ee039c" xlink:to="loc_us-gaap_TradeNamesMember_6a5b0f7b-22b7-49e9-8291-9e592b1ff5f1" xlink:type="arc" order="1"/>
  </link:definitionLink>
</link:linkbase>
