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

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2026 Workiva-->
<!--r:019f905a-2828-7e03-b105-e92efa6be28e,g:8a57f34d-6ce8-4b7c-a154-b1344d471137-->
<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-20260930.xsd#CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnaudited"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnaudited" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_019f905a-28d4-7957-9296-59a41a0b8ebd" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_019f905a-28d4-740a-9e76-8c595ea4fc4d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_019f905a-28d4-7957-9296-59a41a0b8ebd" xlink:to="loc_us-gaap_StatementTable_019f905a-28d4-740a-9e76-8c595ea4fc4d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_019f905a-28d4-7f3c-a251-2d61315488aa" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_019f905a-28d4-740a-9e76-8c595ea4fc4d" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_019f905a-28d4-7f3c-a251-2d61315488aa" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7f3c-a251-2d61315488aa_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019f905a-28d4-7f3c-a251-2d61315488aa" xlink:to="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7f3c-a251-2d61315488aa_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019f905a-28d4-7f3c-a251-2d61315488aa" xlink:to="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_019f905a-28d4-7c7e-bf47-8decf890b79a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:to="loc_us-gaap_CommonStockMember_019f905a-28d4-7c7e-bf47-8decf890b79a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockIncludingAdditionalPaidInCapitalMember_019f905a-28d4-7516-a9d4-b13abed60bfe" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockIncludingAdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:to="loc_us-gaap_CommonStockIncludingAdditionalPaidInCapitalMember_019f905a-28d4-7516-a9d4-b13abed60bfe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_019f905a-28d4-73b1-98b7-d88861c8564b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:to="loc_us-gaap_RetainedEarningsMember_019f905a-28d4-73b1-98b7-d88861c8564b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-28d4-7805-95b0-071a8aaa04f6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-28d4-7805-95b0-071a8aaa04f6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NoncontrollingInterestMember_019f905a-28d4-71d8-976a-ce7a9ca64970" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NoncontrollingInterestMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-28d4-7b0c-a973-9cf9a8d5282c" xlink:to="loc_us-gaap_NoncontrollingInterestMember_019f905a-28d4-71d8-976a-ce7a9ca64970" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019f905a-28d4-7957-9296-59a41a0b8ebd" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_019f905a-28d4-7d1d-9d02-706196153b8a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_019f905a-28d4-7d1d-9d02-706196153b8a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_019f905a-28d4-7e8d-bc5d-76d41754bf8c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_019f905a-28d4-7e8d-bc5d-76d41754bf8c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_019f905a-28d4-7dd9-80bc-5a8f50979b93" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_019f905a-28d4-7dd9-80bc-5a8f50979b93" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_019f905a-28d4-7eac-b5fa-d991a9082a8e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_019f905a-28d4-7dd9-80bc-5a8f50979b93" xlink:to="loc_us-gaap_NetIncomeLoss_019f905a-28d4-7eac-b5fa-d991a9082a8e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_019f905a-28d4-7ce8-8a02-15223690338b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_019f905a-28d4-7dd9-80bc-5a8f50979b93" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPortionAttributableToParent_019f905a-28d4-7ce8-8a02-15223690338b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTax_019f905a-28d4-7c08-ac45-913fb78cb19b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ComprehensiveIncomeNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ComprehensiveIncomeNetOfTaxIncludingPortionAttributableToNoncontrollingInterestAbstract_019f905a-28d4-7dd9-80bc-5a8f50979b93" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTax_019f905a-28d4-7c08-ac45-913fb78cb19b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DividendsCommonStockCash_019f905a-28d4-79ed-be06-a5eb8b8a716e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DividendsCommonStockCash"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_us-gaap_DividendsCommonStockCash_019f905a-28d4-79ed-be06-a5eb8b8a716e" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodShares_019f905a-28d4-7016-8228-f5677e235e21" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockRepurchasedAndRetiredDuringPeriodShares"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodShares_019f905a-28d4-7016-8228-f5677e235e21" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodValue_019f905a-28d4-7848-a55b-862bc3b32628" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockRepurchasedAndRetiredDuringPeriodValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_us-gaap_StockRepurchasedAndRetiredDuringPeriodValue_019f905a-28d4-7848-a55b-862bc3b32628" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_StockRepurchasedAndRetiredDuringPeriodValueExciseTax_019f905a-28d4-7e92-8e58-c93af3bdfa11" xlink:href="csx-20260930.xsd#csx_StockRepurchasedAndRetiredDuringPeriodValueExciseTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_csx_StockRepurchasedAndRetiredDuringPeriodValueExciseTax_019f905a-28d4-7e92-8e58-c93af3bdfa11" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodAndOther_019f905a-28d4-7f22-87f8-18c49b16d519" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodAndOther"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodAndOther_019f905a-28d4-7f22-87f8-18c49b16d519" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_StockIssuedDuringPeriodValueStockOptionsExercisedAndOther_019f905a-28d4-7476-b8dc-dd1e31680f31" xlink:href="csx-20260930.xsd#csx_StockIssuedDuringPeriodValueStockOptionsExercisedAndOther"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019f905a-28d4-7afe-a649-ed60e2f3c082" xlink:to="loc_csx_StockIssuedDuringPeriodValueStockOptionsExercisedAndOther_019f905a-28d4-7476-b8dc-dd1e31680f31" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_019f905a-28d4-73d2-97da-86d044121c00" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_019f905a-297d-75b0-a354-dd351f85ea7d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnauditedParenthetical" xlink:type="simple" xlink:href="csx-20260930.xsd#CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnauditedParenthetical"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CONSOLIDATEDSTATEMENTSOFCHANGESINSHAREHOLDERSEQUITYUnauditedParenthetical" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_019f905a-297d-7124-b98a-a8d0d3449f88" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_019f905a-297d-7f42-b037-9747aec4823d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_019f905a-297d-7124-b98a-a8d0d3449f88" xlink:to="loc_us-gaap_StatementTable_019f905a-297d-7f42-b037-9747aec4823d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297d-7329-a636-7fae628b8ce3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_019f905a-297d-7f42-b037-9747aec4823d" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297d-7329-a636-7fae628b8ce3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019f905a-297d-7329-a636-7fae628b8ce3_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297d-7329-a636-7fae628b8ce3" xlink:to="loc_us-gaap_EquityComponentDomain_019f905a-297d-7329-a636-7fae628b8ce3_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019f905a-297d-7e73-a93a-ed328cfadcdf" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297d-7329-a636-7fae628b8ce3" xlink:to="loc_us-gaap_EquityComponentDomain_019f905a-297d-7e73-a93a-ed328cfadcdf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297d-7d5a-9d82-616226ce3ae5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-297d-7e73-a93a-ed328cfadcdf" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297d-7d5a-9d82-616226ce3ae5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockDividendsPerShareDeclared_019f905a-297d-7d3a-ae95-adb9bae5955b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockDividendsPerShareDeclared"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019f905a-297d-7124-b98a-a8d0d3449f88" xlink:to="loc_us-gaap_CommonStockDividendsPerShareDeclared_019f905a-297d-7d3a-ae95-adb9bae5955b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AociTaxAttributableToParent_019f905a-297d-7098-bb02-5764c5469ae5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AociTaxAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019f905a-297d-7124-b98a-a8d0d3449f88" xlink:to="loc_us-gaap_AociTaxAttributableToParent_019f905a-297d-7098-bb02-5764c5469ae5" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EarningsPerShareScheduleofTotalAverageOutstandingStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#EarningsPerShareScheduleofTotalAverageOutstandingStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EarningsPerShareScheduleofTotalAverageOutstandingStockOptionsExcludedfromDilutedEarningsPerShareCalculationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-709a-9afb-e68edd4a013e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019f905a-297d-7f9f-a998-0670d7bcd63b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-709a-9afb-e68edd4a013e" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019f905a-297d-7f9f-a998-0670d7bcd63b" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019f905a-297d-7e51-8222-7df1f01d0227" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019f905a-297d-7f9f-a998-0670d7bcd63b" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019f905a-297d-7e51-8222-7df1f01d0227" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019f905a-297d-7e51-8222-7df1f01d0227_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019f905a-297d-7e51-8222-7df1f01d0227" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019f905a-297d-7e51-8222-7df1f01d0227_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019f905a-297d-7224-8f97-0a800deef038" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019f905a-297d-7e51-8222-7df1f01d0227" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019f905a-297d-7224-8f97-0a800deef038" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-73d8-aae2-85d5b632edfe" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019f905a-297d-7224-8f97-0a800deef038" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-73d8-aae2-85d5b632edfe" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_019f905a-297d-7a46-b0c9-6c5fd6a33612" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-709a-9afb-e68edd4a013e" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_019f905a-297d-7a46-b0c9-6c5fd6a33612" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EarningsPerShareNarrativeDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#EarningsPerShareNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EarningsPerShareNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-7326-96f4-55afa02ad4d1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019f905a-297d-77f6-9ff5-07f50f4b3633" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-7326-96f4-55afa02ad4d1" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019f905a-297d-77f6-9ff5-07f50f4b3633" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ShareRepurchaseProgramAxis_019f905a-297d-73c9-82e2-4313dc7750c4" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ShareRepurchaseProgramAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019f905a-297d-77f6-9ff5-07f50f4b3633" xlink:to="loc_srt_ShareRepurchaseProgramAxis_019f905a-297d-73c9-82e2-4313dc7750c4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ShareRepurchaseProgramDomain_019f905a-297d-73c9-82e2-4313dc7750c4_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ShareRepurchaseProgramDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ShareRepurchaseProgramAxis_019f905a-297d-73c9-82e2-4313dc7750c4" xlink:to="loc_srt_ShareRepurchaseProgramDomain_019f905a-297d-73c9-82e2-4313dc7750c4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ShareRepurchaseProgramDomain_019f905a-297d-732b-b9bf-4b80b1e7c8ce" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ShareRepurchaseProgramDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ShareRepurchaseProgramAxis_019f905a-297d-73c9-82e2-4313dc7750c4" xlink:to="loc_srt_ShareRepurchaseProgramDomain_019f905a-297d-732b-b9bf-4b80b1e7c8ce" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareRepurchaseProgramOctober2023May2026Member_019f905a-297d-7c68-aefd-472386c20c48" xlink:href="csx-20260930.xsd#csx_ShareRepurchaseProgramOctober2023May2026Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ShareRepurchaseProgramDomain_019f905a-297d-732b-b9bf-4b80b1e7c8ce" xlink:to="loc_csx_ShareRepurchaseProgramOctober2023May2026Member_019f905a-297d-7c68-aefd-472386c20c48" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareRepurchaseProgramOctober2023Member_019f905a-297d-7460-8a6a-cc4c7f09db10" xlink:href="csx-20260930.xsd#csx_ShareRepurchaseProgramOctober2023Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_ShareRepurchaseProgramOctober2023May2026Member_019f905a-297d-7c68-aefd-472386c20c48" xlink:to="loc_csx_ShareRepurchaseProgramOctober2023Member_019f905a-297d-7460-8a6a-cc4c7f09db10" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareRepurchaseProgramMay2026Member_019f905a-297d-7313-9534-91bffd950043" xlink:href="csx-20260930.xsd#csx_ShareRepurchaseProgramMay2026Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_ShareRepurchaseProgramOctober2023May2026Member_019f905a-297d-7c68-aefd-472386c20c48" xlink:to="loc_csx_ShareRepurchaseProgramMay2026Member_019f905a-297d-7313-9534-91bffd950043" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StockRepurchaseProgramAuthorizedAmount1_019f905a-297d-7542-922e-2834068e3413" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_StockRepurchaseProgramAuthorizedAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-7326-96f4-55afa02ad4d1" xlink:to="loc_srt_StockRepurchaseProgramAuthorizedAmount1_019f905a-297d-7542-922e-2834068e3413" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1_019f905a-297d-794d-89fd-5af4add9b699" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-7326-96f4-55afa02ad4d1" xlink:to="loc_us-gaap_StockRepurchaseProgramRemainingAuthorizedRepurchaseAmount1_019f905a-297d-794d-89fd-5af4add9b699" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CommonStockDividendRateIncreasePercentage_019f905a-297d-72bf-b5d9-3623f22e00b2" xlink:href="csx-20260930.xsd#csx_CommonStockDividendRateIncreasePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-7326-96f4-55afa02ad4d1" xlink:to="loc_csx_CommonStockDividendRateIncreasePercentage_019f905a-297d-72bf-b5d9-3623f22e00b2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockDividendsPerShareDeclared_019f905a-297d-7818-983c-f26ccdc6a392" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockDividendsPerShareDeclared"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019f905a-297d-7326-96f4-55afa02ad4d1" xlink:to="loc_us-gaap_CommonStockDividendsPerShareDeclared_019f905a-297d-7818-983c-f26ccdc6a392" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationScheduleofShareBasedCompensationandRelatedIncomeTaxBenefitDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#StockPlansandShareBasedCompensationScheduleofShareBasedCompensationandRelatedIncomeTaxBenefitDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationScheduleofShareBasedCompensationandRelatedIncomeTaxBenefitDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7f39-ac4f-bd5a38360a3c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7eb9-90d9-b7ce40e4cb1e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7f39-ac4f-bd5a38360a3c" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7eb9-90d9-b7ce40e4cb1e" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019f905a-297d-7da2-ab57-d4c5fa26e1d0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7eb9-90d9-b7ce40e4cb1e" xlink:to="loc_us-gaap_AwardTypeAxis_019f905a-297d-7da2-ab57-d4c5fa26e1d0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7da2-ab57-d4c5fa26e1d0_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019f905a-297d-7da2-ab57-d4c5fa26e1d0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7da2-ab57-d4c5fa26e1d0_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019f905a-297d-7da2-ab57-d4c5fa26e1d0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PerformanceSharesMember_019f905a-297d-713a-9835-0fb101ba3090" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PerformanceSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:to="loc_us-gaap_PerformanceSharesMember_019f905a-297d-713a-9835-0fb101ba3090" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019f905a-297d-7f76-aa04-35ccfdea921e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019f905a-297d-7f76-aa04-35ccfdea921e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-760d-bd32-909640c9b007" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-760d-bd32-909640c9b007" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_019f905a-297d-7573-aec1-fb57302a4bfe" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:to="loc_us-gaap_EmployeeStockMember_019f905a-297d-7573-aec1-fb57302a4bfe" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DirectorStockAwardsMember_019f905a-297d-786a-b92e-c030b726ce2a" xlink:href="csx-20260930.xsd#csx_DirectorStockAwardsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7b81-9e3b-41ff9c329b93" xlink:to="loc_csx_DirectorStockAwardsMember_019f905a-297d-786a-b92e-c030b726ce2a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_019f905a-297d-7d43-bd7a-ee69310297ba" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7f39-ac4f-bd5a38360a3c" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_019f905a-297d-7d43-bd7a-ee69310297ba" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationTaxBenefitFromCompensationExpense_019f905a-297d-7bae-bf1b-4dc5c04cc10d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationTaxBenefitFromCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7f39-ac4f-bd5a38360a3c" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationTaxBenefitFromCompensationExpense_019f905a-297d-7bae-bf1b-4dc5c04cc10d" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationScheduleofShareBasedCompensationArrangementsbyShareBasedPaymentAwardDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#StockPlansandShareBasedCompensationScheduleofShareBasedCompensationArrangementsbyShareBasedPaymentAwardDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationScheduleofShareBasedCompensationArrangementsbyShareBasedPaymentAwardDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7098-8fa2-3b13d63aa5ae" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7001-96c2-bcf2f4c4cc6d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7098-8fa2-3b13d63aa5ae" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7001-96c2-bcf2f4c4cc6d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019f905a-297d-7981-bfa7-d29283011db5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7001-96c2-bcf2f4c4cc6d" xlink:to="loc_us-gaap_AwardTypeAxis_019f905a-297d-7981-bfa7-d29283011db5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7981-bfa7-d29283011db5_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019f905a-297d-7981-bfa7-d29283011db5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7981-bfa7-d29283011db5_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-737a-a46a-46f3cbb98662" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019f905a-297d-7981-bfa7-d29283011db5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-737a-a46a-46f3cbb98662" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PerformanceSharesMember_019f905a-297d-71eb-98b2-91541b30151a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PerformanceSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-737a-a46a-46f3cbb98662" xlink:to="loc_us-gaap_PerformanceSharesMember_019f905a-297d-71eb-98b2-91541b30151a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019f905a-297d-7e96-a10f-445dd3c0cda4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-737a-a46a-46f3cbb98662" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019f905a-297d-7e96-a10f-445dd3c0cda4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-7326-a797-97ed4500368b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-737a-a46a-46f3cbb98662" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-7326-a797-97ed4500368b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_019f905a-297d-7a06-8618-66e8c1a015cb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-7001-96c2-bcf2f4c4cc6d" xlink:to="loc_us-gaap_PlanNameAxis_019f905a-297d-7a06-8618-66e8c1a015cb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019f905a-297d-7a06-8618-66e8c1a015cb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_019f905a-297d-7a06-8618-66e8c1a015cb" xlink:to="loc_us-gaap_PlanNameDomain_019f905a-297d-7a06-8618-66e8c1a015cb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019f905a-297d-7625-8386-eedb3e32f0b8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_019f905a-297d-7a06-8618-66e8c1a015cb" xlink:to="loc_us-gaap_PlanNameDomain_019f905a-297d-7625-8386-eedb3e32f0b8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongtermIncentivePlanMember_019f905a-297d-7519-9c06-aa7c9daa1bbf" xlink:href="csx-20260930.xsd#csx_LongtermIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019f905a-297d-7625-8386-eedb3e32f0b8" xlink:to="loc_csx_LongtermIncentivePlanMember_019f905a-297d-7519-9c06-aa7c9daa1bbf" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019f905a-297d-797d-a490-4a6b1cec306a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7098-8fa2-3b13d63aa5ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019f905a-297d-797d-a490-4a6b1cec306a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_019f905a-297d-7e89-b6ff-224b90fa4956" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7098-8fa2-3b13d63aa5ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_019f905a-297d-7e89-b6ff-224b90fa4956" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_019f905a-297d-7ec3-a27e-195dc623ae65" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7098-8fa2-3b13d63aa5ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_019f905a-297d-7ec3-a27e-195dc623ae65" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_019f905a-297d-7c17-ace5-03c4a35176dd" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7098-8fa2-3b13d63aa5ae" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_019f905a-297d-7c17-ace5-03c4a35176dd" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/StockPlansandShareBasedCompensationNarrativeDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#StockPlansandShareBasedCompensationNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/StockPlansandShareBasedCompensationNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-73aa-85c5-50339693937f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-73aa-85c5-50339693937f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019f905a-297d-7d26-bddd-260890c9e71f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-73aa-85c5-50339693937f" xlink:to="loc_us-gaap_AwardTypeAxis_019f905a-297d-7d26-bddd-260890c9e71f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7d26-bddd-260890c9e71f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019f905a-297d-7d26-bddd-260890c9e71f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-7d26-bddd-260890c9e71f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-736a-aef7-c870bf2803ad" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019f905a-297d-7d26-bddd-260890c9e71f" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-736a-aef7-c870bf2803ad" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PerformanceSharesMember_019f905a-297d-77d3-9490-bc44662e511a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PerformanceSharesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-736a-aef7-c870bf2803ad" xlink:to="loc_us-gaap_PerformanceSharesMember_019f905a-297d-77d3-9490-bc44662e511a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-7f67-985b-3e7427225ac6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-736a-aef7-c870bf2803ad" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019f905a-297d-7f67-985b-3e7427225ac6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019f905a-297d-7c0b-898e-db581bc56985" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019f905a-297d-736a-aef7-c870bf2803ad" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019f905a-297d-7c0b-898e-db581bc56985" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_019f905a-297d-7dde-8f71-75ce82d0b6af" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019f905a-297d-73aa-85c5-50339693937f" xlink:to="loc_us-gaap_PlanNameAxis_019f905a-297d-7dde-8f71-75ce82d0b6af" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019f905a-297d-7dde-8f71-75ce82d0b6af_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_019f905a-297d-7dde-8f71-75ce82d0b6af" xlink:to="loc_us-gaap_PlanNameDomain_019f905a-297d-7dde-8f71-75ce82d0b6af_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019f905a-297d-7e05-a2ea-4717a11920af" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_019f905a-297d-7dde-8f71-75ce82d0b6af" xlink:to="loc_us-gaap_PlanNameDomain_019f905a-297d-7e05-a2ea-4717a11920af" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongtermIncentivePlanMember_019f905a-297d-74bf-a006-c8ac0cff256d" xlink:href="csx-20260930.xsd#csx_LongtermIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019f905a-297d-7e05-a2ea-4717a11920af" xlink:to="loc_csx_LongtermIncentivePlanMember_019f905a-297d-74bf-a006-c8ac0cff256d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_019f905a-297d-7623-ad9d-498818e9fa71" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_019f905a-297d-7623-ad9d-498818e9fa71" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsReturnOnInvestedCapitalWeight_019f905a-297d-7e5c-9774-c894614d2c83" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsReturnOnInvestedCapitalWeight"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsReturnOnInvestedCapitalWeight_019f905a-297d-7e5c-9774-c894614d2c83" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsRelativeTotalShareholderReturnWeight_019f905a-297d-7f05-8794-9a488851e49f" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsRelativeTotalShareholderReturnWeight"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsRelativeTotalShareholderReturnWeight_019f905a-297d-7f05-8794-9a488851e49f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageGrantValue_019f905a-297d-7f9f-bc3d-e8b11e4078d6" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageGrantValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsPayoutPercentageGrantValue_019f905a-297d-7f9f-bc3d-e8b11e4078d6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumReturnOnInvestedCapitalWeight_019f905a-297d-76a4-a84d-c72eed1dfc72" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumReturnOnInvestedCapitalWeight"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumReturnOnInvestedCapitalWeight_019f905a-297d-76a4-a84d-c72eed1dfc72" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumRelativeTotalShareholderReturnWeight_019f905a-297d-78d7-8087-afa522b5aba3" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumRelativeTotalShareholderReturnWeight"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumRelativeTotalShareholderReturnWeight_019f905a-297d-78d7-8087-afa522b5aba3" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumPayoutPercentage_019f905a-297d-70c2-aaa7-69bb87d263e1" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumPayoutPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsMaximumPayoutPercentage_019f905a-297d-70c2-aaa7-69bb87d263e1" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_019f905a-297d-7b86-b773-103bdae05366" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_019f905a-297d-7b86-b773-103bdae05366" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfEquivalentSharesOfCommonStockPerUnit_019f905a-297d-7048-8662-a356321d6bfb" xlink:href="csx-20260930.xsd#csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfEquivalentSharesOfCommonStockPerUnit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019f905a-297d-7647-8484-a5a8af6205b4" xlink:to="loc_csx_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfEquivalentSharesOfCommonStockPerUnit_019f905a-297d-7048-8662-a356321d6bfb" xlink:type="arc" order="8"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesScheduleofBalanceSheetPresentationDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#CasualtyEnvironmentalandOtherReservesScheduleofBalanceSheetPresentationDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesScheduleofBalanceSheetPresentationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_csx_ContingenciesLineItems_019f905a-297d-78d8-9dc4-d0cdf943db99" xlink:href="csx-20260930.xsd#csx_ContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ContingenciesTable_019f905a-297d-7577-89f5-0d6752c880b2" xlink:href="csx-20260930.xsd#csx_ContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_csx_ContingenciesLineItems_019f905a-297d-78d8-9dc4-d0cdf943db99" xlink:to="loc_csx_ContingenciesTable_019f905a-297d-7577-89f5-0d6752c880b2" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7b25-80a4-13b2117ffca2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_csx_ContingenciesTable_019f905a-297d-7577-89f5-0d6752c880b2" xlink:to="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7b25-80a4-13b2117ffca2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7b25-80a4-13b2117ffca2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7b25-80a4-13b2117ffca2" xlink:to="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7b25-80a4-13b2117ffca2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7af2-80b8-4ad68e0ec95a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7b25-80a4-13b2117ffca2" xlink:to="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7af2-80b8-4ad68e0ec95a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalCasualtyMember_019f905a-297d-75db-8783-8862ecebce3c" xlink:href="csx-20260930.xsd#csx_TotalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7af2-80b8-4ad68e0ec95a" xlink:to="loc_csx_TotalCasualtyMember_019f905a-297d-75db-8783-8862ecebce3c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_PersonalInjuryCasualtyMember_019f905a-297d-77cf-a094-2e14fc83141b" xlink:href="csx-20260930.xsd#csx_PersonalInjuryCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalCasualtyMember_019f905a-297d-75db-8783-8862ecebce3c" xlink:to="loc_csx_PersonalInjuryCasualtyMember_019f905a-297d-77cf-a094-2e14fc83141b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OccupationalCasualtyMember_019f905a-297d-766d-99ae-30bcedaf11a3" xlink:href="csx-20260930.xsd#csx_OccupationalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalCasualtyMember_019f905a-297d-75db-8783-8862ecebce3c" xlink:to="loc_csx_OccupationalCasualtyMember_019f905a-297d-766d-99ae-30bcedaf11a3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EnvironmentalIssueMember_019f905a-297d-71a6-bb43-556e0af3ece3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EnvironmentalIssueMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7af2-80b8-4ad68e0ec95a" xlink:to="loc_us-gaap_EnvironmentalIssueMember_019f905a-297d-71a6-bb43-556e0af3ece3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OtherReservesMember_019f905a-297d-7e64-b491-f81d29b20057" xlink:href="csx-20260930.xsd#csx_OtherReservesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7af2-80b8-4ad68e0ec95a" xlink:to="loc_csx_OtherReservesMember_019f905a-297d-7e64-b491-f81d29b20057" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualCarryingValueCurrent_019f905a-297d-7f44-8a56-6652e3d7df34" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyAccrualCarryingValueCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_ContingenciesLineItems_019f905a-297d-78d8-9dc4-d0cdf943db99" xlink:to="loc_us-gaap_LossContingencyAccrualCarryingValueCurrent_019f905a-297d-7f44-8a56-6652e3d7df34" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualCarryingValueNoncurrent_019f905a-297d-7d37-a7d3-0b58f3e54b13" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyAccrualCarryingValueNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_ContingenciesLineItems_019f905a-297d-78d8-9dc4-d0cdf943db99" xlink:to="loc_us-gaap_LossContingencyAccrualCarryingValueNoncurrent_019f905a-297d-7d37-a7d3-0b58f3e54b13" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualAtCarryingValue_019f905a-297d-7ad6-9cbc-3002a487ae06" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyAccrualAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_ContingenciesLineItems_019f905a-297d-78d8-9dc4-d0cdf943db99" xlink:to="loc_us-gaap_LossContingencyAccrualAtCarryingValue_019f905a-297d-7ad6-9cbc-3002a487ae06" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesNarrativeDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#CasualtyEnvironmentalandOtherReservesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CasualtyEnvironmentalandOtherReservesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_019f905a-297d-78d1-b2a5-d406099585c0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesTable_019f905a-297d-744e-a39d-7a8555d8e18c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297d-78d1-b2a5-d406099585c0" xlink:to="loc_us-gaap_LossContingenciesTable_019f905a-297d-744e-a39d-7a8555d8e18c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7cdc-9725-8fcd6c476586" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_019f905a-297d-744e-a39d-7a8555d8e18c" xlink:to="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7cdc-9725-8fcd6c476586" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7cdc-9725-8fcd6c476586_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7cdc-9725-8fcd6c476586" xlink:to="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-7cdc-9725-8fcd6c476586_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-77d7-914e-419b6e171378" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297d-7cdc-9725-8fcd6c476586" xlink:to="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-77d7-914e-419b6e171378" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalCasualtyMember_019f905a-297d-7999-8d68-b7c9a52680a1" xlink:href="csx-20260930.xsd#csx_TotalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-77d7-914e-419b6e171378" xlink:to="loc_csx_TotalCasualtyMember_019f905a-297d-7999-8d68-b7c9a52680a1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EnvironmentalIssueMember_019f905a-297d-7977-9792-dcf84638058b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EnvironmentalIssueMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_019f905a-297d-77d7-914e-419b6e171378" xlink:to="loc_us-gaap_EnvironmentalIssueMember_019f905a-297d-7977-9792-dcf84638058b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SelfInsuredRetention_019f905a-297d-7de3-b8d0-60d4721f1bc3" xlink:href="csx-20260930.xsd#csx_SelfInsuredRetention"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297d-78d1-b2a5-d406099585c0" xlink:to="loc_csx_SelfInsuredRetention_019f905a-297d-7de3-b8d0-60d4721f1bc3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyNumberOfClaimsExpectedToExceedSelfInsuredRetentionAmount_019f905a-297d-73cc-b032-c870f5886f62" xlink:href="csx-20260930.xsd#csx_LossContingencyNumberOfClaimsExpectedToExceedSelfInsuredRetentionAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297d-78d1-b2a5-d406099585c0" xlink:to="loc_csx_LossContingencyNumberOfClaimsExpectedToExceedSelfInsuredRetentionAmount_019f905a-297d-73cc-b032-c870f5886f62" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfImpairedSites_019f905a-297d-70f1-92a7-5fe10c5cf18b" xlink:href="csx-20260930.xsd#csx_NumberOfImpairedSites"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297d-78d1-b2a5-d406099585c0" xlink:to="loc_csx_NumberOfImpairedSites_019f905a-297d-70f1-92a7-5fe10c5cf18b" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/CommitmentsandContingenciesDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#CommitmentsandContingenciesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/CommitmentsandContingenciesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherCommitmentsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297e-7a98-a5dc-a403414cced5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:to="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297e-7a98-a5dc-a403414cced5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_019f905a-297e-7a98-a5dc-a403414cced5_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297e-7a98-a5dc-a403414cced5" xlink:to="loc_us-gaap_LossContingencyNatureDomain_019f905a-297e-7a98-a5dc-a403414cced5_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNatureDomain_019f905a-297e-77e4-bcf8-fc4696c91bc6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNatureDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LossContingenciesByNatureOfContingencyAxis_019f905a-297e-7a98-a5dc-a403414cced5" xlink:to="loc_us-gaap_LossContingencyNatureDomain_019f905a-297e-77e4-bcf8-fc4696c91bc6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalCasualtyMember_019f905a-297e-75f3-8a24-65ca3f798502" xlink:href="csx-20260930.xsd#csx_TotalCasualtyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingencyNatureDomain_019f905a-297e-77e4-bcf8-fc4696c91bc6" xlink:to="loc_csx_TotalCasualtyMember_019f905a-297e-75f3-8a24-65ca3f798502" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_019f905a-297e-7b72-a267-a68e4d1b732c" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:to="loc_srt_RangeAxis_019f905a-297e-7b72-a267-a68e4d1b732c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_019f905a-297e-7b72-a267-a68e4d1b732c_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_019f905a-297e-7b72-a267-a68e4d1b732c" xlink:to="loc_srt_RangeMember_019f905a-297e-7b72-a267-a68e4d1b732c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_019f905a-297e-749e-8452-4c2d5ebabc57" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_019f905a-297e-7b72-a267-a68e4d1b732c" xlink:to="loc_srt_RangeMember_019f905a-297e-749e-8452-4c2d5ebabc57" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_019f905a-297e-7011-b367-787dfb75a6cc" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_019f905a-297e-749e-8452-4c2d5ebabc57" xlink:to="loc_srt_MinimumMember_019f905a-297e-7011-b367-787dfb75a6cc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_019f905a-297e-72d4-ab9c-103c14a8cbcb" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_019f905a-297e-749e-8452-4c2d5ebabc57" xlink:to="loc_srt_MaximumMember_019f905a-297e-72d4-ab9c-103c14a8cbcb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationStatusAxis_019f905a-297e-7922-85c2-551ce40f0572" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LitigationStatusAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:to="loc_us-gaap_LitigationStatusAxis_019f905a-297e-7922-85c2-551ce40f0572" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationStatusDomain_019f905a-297e-7922-85c2-551ce40f0572_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LitigationStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LitigationStatusAxis_019f905a-297e-7922-85c2-551ce40f0572" xlink:to="loc_us-gaap_LitigationStatusDomain_019f905a-297e-7922-85c2-551ce40f0572_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationStatusDomain_019f905a-297e-7fb9-9542-96ff3759279c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LitigationStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LitigationStatusAxis_019f905a-297e-7922-85c2-551ce40f0572" xlink:to="loc_us-gaap_LitigationStatusDomain_019f905a-297e-7fb9-9542-96ff3759279c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PendingLitigationMember_019f905a-297e-7061-b120-5959f6a10b34" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PendingLitigationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LitigationStatusDomain_019f905a-297e-7fb9-9542-96ff3759279c" xlink:to="loc_us-gaap_PendingLitigationMember_019f905a-297e-7061-b120-5959f6a10b34" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_LitigationCaseAxis_019f905a-297e-7be8-9b80-06766988c281" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_LitigationCaseAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:to="loc_srt_LitigationCaseAxis_019f905a-297e-7be8-9b80-06766988c281" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_LitigationCaseTypeDomain_019f905a-297e-7be8-9b80-06766988c281_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_LitigationCaseTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_LitigationCaseAxis_019f905a-297e-7be8-9b80-06766988c281" xlink:to="loc_srt_LitigationCaseTypeDomain_019f905a-297e-7be8-9b80-06766988c281_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_LitigationCaseTypeDomain_019f905a-297e-77eb-988a-cbda583f7a0d" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_LitigationCaseTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_LitigationCaseAxis_019f905a-297e-7be8-9b80-06766988c281" xlink:to="loc_srt_LitigationCaseTypeDomain_019f905a-297e-77eb-988a-cbda583f7a0d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelSurchargeAntitrustLitigationMember_019f905a-297e-7b4c-a9cf-32eff00ad89e" xlink:href="csx-20260930.xsd#csx_FuelSurchargeAntitrustLitigationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_LitigationCaseTypeDomain_019f905a-297e-77eb-988a-cbda583f7a0d" xlink:to="loc_csx_FuelSurchargeAntitrustLitigationMember_019f905a-297e-7b4c-a9cf-32eff00ad89e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_EnvironmentalLitigationMember_019f905a-297e-7a7c-83d1-dc6b98900e0a" xlink:href="csx-20260930.xsd#csx_EnvironmentalLitigationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_LitigationCaseTypeDomain_019f905a-297e-77eb-988a-cbda583f7a0d" xlink:to="loc_csx_EnvironmentalLitigationMember_019f905a-297e-7a7c-83d1-dc6b98900e0a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_LegalEntityAxis_019f905a-297e-7ecd-abb1-215a115e2735" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_OtherCommitmentsTable_019f905a-297e-72b2-81ea-30245850482d" xlink:to="loc_dei_LegalEntityAxis_019f905a-297e-7ecd-abb1-215a115e2735" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityDomain_019f905a-297e-7ecd-abb1-215a115e2735_default" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_dei_LegalEntityAxis_019f905a-297e-7ecd-abb1-215a115e2735" xlink:to="loc_dei_EntityDomain_019f905a-297e-7ecd-abb1-215a115e2735_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityDomain_019f905a-297e-7a32-b4c6-0cb8e0126fd1" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_dei_LegalEntityAxis_019f905a-297e-7ecd-abb1-215a115e2735" xlink:to="loc_dei_EntityDomain_019f905a-297e-7a32-b4c6-0cb8e0126fd1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OtherDefendantsMember_019f905a-297e-75b0-b347-4e8f6dbb222b" xlink:href="csx-20260930.xsd#csx_OtherDefendantsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityDomain_019f905a-297e-7a32-b4c6-0cb8e0126fd1" xlink:to="loc_csx_OtherDefendantsMember_019f905a-297e-75b0-b347-4e8f6dbb222b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OccidentalPetroleumCorporationMember_019f905a-297e-7c00-97eb-18af767fb521" xlink:href="csx-20260930.xsd#csx_OccidentalPetroleumCorporationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityDomain_019f905a-297e-7a32-b4c6-0cb8e0126fd1" xlink:to="loc_csx_OccidentalPetroleumCorporationMember_019f905a-297e-7c00-97eb-18af767fb521" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CasualtyAndNonCatastrophicPropertyDeductible_019f905a-297e-7d93-ac4b-1de7492cdfcf" xlink:href="csx-20260930.xsd#csx_CasualtyAndNonCatastrophicPropertyDeductible"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_CasualtyAndNonCatastrophicPropertyDeductible_019f905a-297e-7d93-ac4b-1de7492cdfcf" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CasualtyAndCatastrophicPropertyDeductible_019f905a-297e-7ec8-b1af-909de4ec810f" xlink:href="csx-20260930.xsd#csx_CasualtyAndCatastrophicPropertyDeductible"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_CasualtyAndCatastrophicPropertyDeductible_019f905a-297e-7ec8-b1af-909de4ec810f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SelfInsuredRetention_019f905a-297e-778d-8c10-7bdda0481c73" xlink:href="csx-20260930.xsd#csx_SelfInsuredRetention"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_SelfInsuredRetention_019f905a-297e-778d-8c10-7bdda0481c73" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyEstimateOfPossibleLoss_019f905a-297e-7dc2-bfea-b0b88ac73557" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyEstimateOfPossibleLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_us-gaap_LossContingencyEstimateOfPossibleLoss_019f905a-297e-7dc2-bfea-b0b88ac73557" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyClaimsFiledNumberOfEntities_019f905a-297e-71b0-90c2-aea3e28ba8f4" xlink:href="csx-20260930.xsd#csx_LossContingencyClaimsFiledNumberOfEntities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_LossContingencyClaimsFiledNumberOfEntities_019f905a-297e-71b0-90c2-aea3e28ba8f4" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfMilesPertainingToPassaicRiverTidalReachRequiredToBeStudiedByEnvironmentalProtectionAgency_019f905a-297e-72c5-92ec-3914975677ed" xlink:href="csx-20260930.xsd#csx_NumberOfMilesPertainingToPassaicRiverTidalReachRequiredToBeStudiedByEnvironmentalProtectionAgency"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_NumberOfMilesPertainingToPassaicRiverTidalReachRequiredToBeStudiedByEnvironmentalProtectionAgency_019f905a-297e-72c5-92ec-3914975677ed" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SiteContingencyNumberOfLowerMilesUnderStudy_019f905a-297e-7993-a591-a5135460c828" xlink:href="csx-20260930.xsd#csx_SiteContingencyNumberOfLowerMilesUnderStudy"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_SiteContingencyNumberOfLowerMilesUnderStudy_019f905a-297e-7993-a591-a5135460c828" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SiteContingencyNumberOfUpperMilesUnderStudy_019f905a-297e-76e4-b11d-f91e08e1427e" xlink:href="csx-20260930.xsd#csx_SiteContingencyNumberOfUpperMilesUnderStudy"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_SiteContingencyNumberOfUpperMilesUnderStudy_019f905a-297e-76e4-b11d-f91e08e1427e" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SiteContingencyNumberOfPartiesLiable_019f905a-297e-787f-8b6e-d0f6e0767a1c" xlink:href="csx-20260930.xsd#csx_SiteContingencyNumberOfPartiesLiable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_SiteContingencyNumberOfPartiesLiable_019f905a-297e-787f-8b6e-d0f6e0767a1c" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SiteContingencyNumberOfPartiesParticipatingInModifiedCD_019f905a-297e-7047-a6c5-ee9af87e5bd5" xlink:href="csx-20260930.xsd#csx_SiteContingencyNumberOfPartiesParticipatingInModifiedCD"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_SiteContingencyNumberOfPartiesParticipatingInModifiedCD_019f905a-297e-7047-a6c5-ee9af87e5bd5" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LitigationSettlementAmountAwardedToOtherParty_019f905a-297e-7d36-97fb-d90d6eaba191" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LitigationSettlementAmountAwardedToOtherParty"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_us-gaap_LitigationSettlementAmountAwardedToOtherParty_019f905a-297e-7d36-97fb-d90d6eaba191" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyNumberOfEntities_019f905a-297e-72e9-9b3c-35627e6e124a" xlink:href="csx-20260930.xsd#csx_LossContingencyNumberOfEntities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_LossContingencyNumberOfEntities_019f905a-297e-72e9-9b3c-35627e6e124a" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyCleanupWork_01a1102b-4951-7fee-ad0e-6f4c4bb40344" xlink:href="csx-20260930.xsd#csx_LossContingencyCleanupWork"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_LossContingencyCleanupWork_01a1102b-4951-7fee-ad0e-6f4c4bb40344" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyBaselineSamplingNumberOfMiles_01a1102c-fa75-7acd-8261-5aa9579db5b0" xlink:href="csx-20260930.xsd#csx_LossContingencyBaselineSamplingNumberOfMiles"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_LossContingencyBaselineSamplingNumberOfMiles_01a1102c-fa75-7acd-8261-5aa9579db5b0" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LossContingencyRequiredFinancialAssuranceToBeSecured_01a1102f-121a-7ddb-a11a-ee19c2ab826b" xlink:href="csx-20260930.xsd#csx_LossContingencyRequiredFinancialAssuranceToBeSecured"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_csx_LossContingencyRequiredFinancialAssuranceToBeSecured_01a1102f-121a-7ddb-a11a-ee19c2ab826b" xlink:type="arc" order="14"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyNumberOfDefendants_019f905a-297e-7368-890f-19dd13730141" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LossContingencyNumberOfDefendants"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_019f905a-297e-7df1-ab09-5b04ab106c5b" xlink:to="loc_us-gaap_LossContingencyNumberOfDefendants_019f905a-297e-7368-890f-19dd13730141" xlink:type="arc" order="15"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/EmployeeBenefitPlansScheduleofComponentsofNetPeriodicBenefitCostsDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#EmployeeBenefitPlansScheduleofComponentsofNetPeriodicBenefitCostsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/EmployeeBenefitPlansScheduleofComponentsofNetPeriodicBenefitCostsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_019f905a-297e-7ac1-b3c8-671ac2f362e4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanDisclosureLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_019f905a-297e-79d0-a82b-95479d9efa09" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_019f905a-297e-7ac1-b3c8-671ac2f362e4" xlink:to="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_019f905a-297e-79d0-a82b-95479d9efa09" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeAxis_019f905a-297e-7615-936b-130a73db021d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetirementPlanTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfDefinedBenefitPlansDisclosuresTable_019f905a-297e-79d0-a82b-95479d9efa09" xlink:to="loc_us-gaap_RetirementPlanTypeAxis_019f905a-297e-7615-936b-130a73db021d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeDomain_019f905a-297e-7615-936b-130a73db021d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetirementPlanTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RetirementPlanTypeAxis_019f905a-297e-7615-936b-130a73db021d" xlink:to="loc_us-gaap_RetirementPlanTypeDomain_019f905a-297e-7615-936b-130a73db021d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetirementPlanTypeDomain_019f905a-297e-725d-ba38-5e1f13121620" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetirementPlanTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RetirementPlanTypeAxis_019f905a-297e-7615-936b-130a73db021d" xlink:to="loc_us-gaap_RetirementPlanTypeDomain_019f905a-297e-725d-ba38-5e1f13121620" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PensionPlansDefinedBenefitMember_019f905a-297e-776c-bb64-d439949e4a54" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PensionPlansDefinedBenefitMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RetirementPlanTypeDomain_019f905a-297e-725d-ba38-5e1f13121620" xlink:to="loc_us-gaap_PensionPlansDefinedBenefitMember_019f905a-297e-776c-bb64-d439949e4a54" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanDisclosureLineItems_019f905a-297e-7ac1-b3c8-671ac2f362e4" xlink:to="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanServiceCost_019f905a-297e-79fe-9ba3-ab34d59c23a0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanServiceCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:to="loc_us-gaap_DefinedBenefitPlanServiceCost_019f905a-297e-79fe-9ba3-ab34d59c23a0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanInterestCost_019f905a-297e-764d-be5f-4bd9899aa2c9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanInterestCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:to="loc_us-gaap_DefinedBenefitPlanInterestCost_019f905a-297e-764d-be5f-4bd9899aa2c9" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets_019f905a-297e-78e3-be2b-89cb30a59c20" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:to="loc_us-gaap_DefinedBenefitPlanExpectedReturnOnPlanAssets_019f905a-297e-78e3-be2b-89cb30a59c20" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses_019f905a-297e-7c96-9eee-c538801accdc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:to="loc_us-gaap_DefinedBenefitPlanAmortizationOfGainsLosses_019f905a-297e-7c96-9eee-c538801accdc" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetPeriodicDefinedBenefitsExpenseReversalOfExpenseExcludingServiceCostComponent_019f905a-297e-7bb9-b601-990dbe0df15a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetPeriodicDefinedBenefitsExpenseReversalOfExpenseExcludingServiceCostComponent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:to="loc_us-gaap_NetPeriodicDefinedBenefitsExpenseReversalOfExpenseExcludingServiceCostComponent_019f905a-297e-7bb9-b601-990dbe0df15a" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost_019f905a-297e-75d9-b3be-45f2abce9eb6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCostAbstract_019f905a-297e-7b77-bcc6-64194d8136eb" xlink:to="loc_us-gaap_DefinedBenefitPlanNetPeriodicBenefitCost_019f905a-297e-75d9-b3be-45f2abce9eb6" xlink:type="arc" order="6"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsScheduleofActivityRelatedtoLongTermDebtDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#DebtandCreditAgreementsScheduleofActivityRelatedtoLongTermDebtDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsScheduleofActivityRelatedtoLongTermDebtDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_019f905a-297e-7745-889f-15c04506ece5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_019f905a-297e-77cb-b508-0cdee085845c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019f905a-297e-7745-889f-15c04506ece5" xlink:to="loc_us-gaap_DebtInstrumentTable_019f905a-297e-77cb-b508-0cdee085845c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DebtTypeAxis_019f905a-297e-7415-b9e3-69481e947756" xlink:href="csx-20260930.xsd#csx_DebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019f905a-297e-77cb-b508-0cdee085845c" xlink:to="loc_csx_DebtTypeAxis_019f905a-297e-7415-b9e3-69481e947756" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DebtTypeDomain_019f905a-297e-7415-b9e3-69481e947756_default" xlink:href="csx-20260930.xsd#csx_DebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_csx_DebtTypeAxis_019f905a-297e-7415-b9e3-69481e947756" xlink:to="loc_csx_DebtTypeDomain_019f905a-297e-7415-b9e3-69481e947756_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DebtTypeDomain_019f905a-297e-7939-8c68-2a07cdafa443" xlink:href="csx-20260930.xsd#csx_DebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_csx_DebtTypeAxis_019f905a-297e-7415-b9e3-69481e947756" xlink:to="loc_csx_DebtTypeDomain_019f905a-297e-7939-8c68-2a07cdafa443" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CurrentLiabilitiesMember_019f905a-297e-76c8-9897-15dbd9bc7d07" xlink:href="csx-20260930.xsd#csx_CurrentLiabilitiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_DebtTypeDomain_019f905a-297e-7939-8c68-2a07cdafa443" xlink:to="loc_csx_CurrentLiabilitiesMember_019f905a-297e-76c8-9897-15dbd9bc7d07" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongTermLiabilitiesMember_019f905a-297e-7163-b625-8e69e148a249" xlink:href="csx-20260930.xsd#csx_LongTermLiabilitiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_DebtTypeDomain_019f905a-297e-7939-8c68-2a07cdafa443" xlink:to="loc_csx_LongTermLiabilitiesMember_019f905a-297e-7163-b625-8e69e148a249" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:href="csx-20260930.xsd#csx_MovementDebtInstrumentsRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_019f905a-297e-7745-889f-15c04506ece5" xlink:to="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent_019f905a-297e-712a-b526-323e8e3b9245" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:to="loc_us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent_019f905a-297e-712a-b526-323e8e3b9245" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligations_019f905a-297e-7cdf-824f-1c23a63ce284" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligations"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:to="loc_us-gaap_LongTermDebtAndCapitalLeaseObligations_019f905a-297e-7cdf-824f-1c23a63ce284" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtAndCapitalLeaseObligations_019f905a-297e-75a9-818d-a364e35bdfb0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtAndCapitalLeaseObligations"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:to="loc_us-gaap_DebtAndCapitalLeaseObligations_019f905a-297e-75a9-818d-a364e35bdfb0" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RepaymentsOfLongTermDebt_019f905a-297e-7016-85a3-75fa64d51085" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RepaymentsOfLongTermDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:to="loc_us-gaap_RepaymentsOfLongTermDebt_019f905a-297e-7016-85a3-75fa64d51085" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_LongTermDebtIncreaseDecreaseDueToReclassifications_019f905a-297e-7cca-926c-8f0bb195676d" xlink:href="csx-20260930.xsd#csx_LongTermDebtIncreaseDecreaseDueToReclassifications"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:to="loc_csx_LongTermDebtIncreaseDecreaseDueToReclassifications_019f905a-297e-7cca-926c-8f0bb195676d" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AmortizationOfDebtDiscountPremium_019f905a-297e-7351-86ac-9e50c395c767" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AmortizationOfDebtDiscountPremium"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_MovementDebtInstrumentsRollForward_019f905a-297e-7a85-bb5f-8261c7393390" xlink:to="loc_us-gaap_AmortizationOfDebtDiscountPremium_019f905a-297e-7351-86ac-9e50c395c767" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent_019f905a-297e-75b9-9671-8c69b94a23df" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligationsCurrent"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebtAndCapitalLeaseObligations_019f905a-297e-758f-aeb7-e7a53266b2a2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LongTermDebtAndCapitalLeaseObligations"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtAndCapitalLeaseObligations_019f905a-297e-774d-94ec-c6177dc3120d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtAndCapitalLeaseObligations"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsNarrativeDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#DebtandCreditAgreementsNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LineOfCreditFacilityLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LineOfCreditFacilityTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-7e69-b1cd-c1d4396421fa" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:to="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-7e69-b1cd-c1d4396421fa" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-7e69-b1cd-c1d4396421fa_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-7e69-b1cd-c1d4396421fa" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-7e69-b1cd-c1d4396421fa_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-764e-9990-818cda5ce826" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-7e69-b1cd-c1d4396421fa" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-764e-9990-818cda5ce826" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2055Member_019f905a-297e-7980-b4f9-a2537766b1c8" xlink:href="csx-20260930.xsd#csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2055Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-764e-9990-818cda5ce826" xlink:to="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2055Member_019f905a-297e-7980-b4f9-a2537766b1c8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SevenFixedToFloatingInterestRateSwapsMember_019f905a-297e-764c-8d68-1b6654d815eb" xlink:href="csx-20260930.xsd#csx_SevenFixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-764e-9990-818cda5ce826" xlink:to="loc_csx_SevenFixedToFloatingInterestRateSwapsMember_019f905a-297e-764c-8d68-1b6654d815eb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_A325NotesDue2027Member_019f905a-297e-7e40-ba4a-b4344ca01855" xlink:href="csx-20260930.xsd#csx_A325NotesDue2027Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-764e-9990-818cda5ce826" xlink:to="loc_csx_A325NotesDue2027Member_019f905a-297e-7e40-ba4a-b4344ca01855" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_UnsecuredRevolvingCreditFacilityDue2028Member_019f905a-297e-7c43-a26a-d3638e6d4266" xlink:href="csx-20260930.xsd#csx_UnsecuredRevolvingCreditFacilityDue2028Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-764e-9990-818cda5ce826" xlink:to="loc_csx_UnsecuredRevolvingCreditFacilityDue2028Member_019f905a-297e-7c43-a26a-d3638e6d4266" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-70d6-8e12-e34fa1fb71f2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-70d6-8e12-e34fa1fb71f2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-70d6-8e12-e34fa1fb71f2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-70d6-8e12-e34fa1fb71f2" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-70d6-8e12-e34fa1fb71f2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-70d6-8e12-e34fa1fb71f2" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2055Member_019f905a-297e-7068-ae45-79d0d7f764ef" xlink:href="csx-20260930.xsd#csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2055Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:to="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2055Member_019f905a-297e-7068-ae45-79d0d7f764ef" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_SevenFixedToFloatingInterestRateSwapsMember_019f905a-297e-7f3d-81bc-9fd67f0d29dd" xlink:href="csx-20260930.xsd#csx_SevenFixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:to="loc_csx_SevenFixedToFloatingInterestRateSwapsMember_019f905a-297e-7f3d-81bc-9fd67f0d29dd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsMember_019f905a-297e-7e09-8632-15a4f4ac736b" xlink:href="csx-20260930.xsd#csx_TwoSeparateFixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:to="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsMember_019f905a-297e-7e09-8632-15a4f4ac736b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FiveSeparateFixedToFloatingInterestRateSwapsMember_019f905a-297e-7254-af6a-3be0c6ef7b30" xlink:href="csx-20260930.xsd#csx_FiveSeparateFixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:to="loc_csx_FiveSeparateFixedToFloatingInterestRateSwapsMember_019f905a-297e-7254-af6a-3be0c6ef7b30" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2032Member_019f905a-297e-7805-b47e-0b76da6233f7" xlink:href="csx-20260930.xsd#csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2032Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:to="loc_csx_TwoSeparateFixedToFloatingInterestRateSwapsDue2032Member_019f905a-297e-7805-b47e-0b76da6233f7" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestRateSwapMember_019f905a-297e-7559-82c8-52653021dcd8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-782a-8e23-9d13a2d00559" xlink:to="loc_us-gaap_InterestRateSwapMember_019f905a-297e-7559-82c8-52653021dcd8" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-712c-9111-3f03a23283e9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:to="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-712c-9111-3f03a23283e9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-712c-9111-3f03a23283e9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-712c-9111-3f03a23283e9" xlink:to="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-712c-9111-3f03a23283e9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-724f-b807-885e5288eb3c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-712c-9111-3f03a23283e9" xlink:to="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-724f-b807-885e5288eb3c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueHedgingMember_019f905a-297e-7698-8655-0b136e451aa6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-724f-b807-885e5288eb3c" xlink:to="loc_us-gaap_FairValueHedgingMember_019f905a-297e-7698-8655-0b136e451aa6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashFlowHedgingMember_019f905a-297e-7a0f-8937-970be161a38f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashFlowHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-724f-b807-885e5288eb3c" xlink:to="loc_us-gaap_CashFlowHedgingMember_019f905a-297e-7a0f-8937-970be161a38f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-73dc-8aeb-9f974f8b4516" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:to="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-73dc-8aeb-9f974f8b4516" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-73dc-8aeb-9f974f8b4516_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-73dc-8aeb-9f974f8b4516" xlink:to="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-73dc-8aeb-9f974f8b4516_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-7989-a533-01d0ae75c476" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-73dc-8aeb-9f974f8b4516" xlink:to="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-7989-a533-01d0ae75c476" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DesignatedAsHedgingInstrumentMember_019f905a-297e-7990-9c1f-13efc3d8341b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DesignatedAsHedgingInstrumentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-7989-a533-01d0ae75c476" xlink:to="loc_us-gaap_DesignatedAsHedgingInstrumentMember_019f905a-297e-7990-9c1f-13efc3d8341b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityAxis_019f905a-297e-7d82-a3fa-8ba5bf65de8a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CreditFacilityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_019f905a-297e-7f86-a266-67e7c9ea338a" xlink:to="loc_us-gaap_CreditFacilityAxis_019f905a-297e-7d82-a3fa-8ba5bf65de8a" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_019f905a-297e-7d82-a3fa-8ba5bf65de8a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CreditFacilityAxis_019f905a-297e-7d82-a3fa-8ba5bf65de8a" xlink:to="loc_us-gaap_CreditFacilityDomain_019f905a-297e-7d82-a3fa-8ba5bf65de8a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_019f905a-297e-7001-9e2c-9a40a9ed4433" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CreditFacilityAxis_019f905a-297e-7d82-a3fa-8ba5bf65de8a" xlink:to="loc_us-gaap_CreditFacilityDomain_019f905a-297e-7001-9e2c-9a40a9ed4433" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevolvingCreditFacilityMember_019f905a-297e-7f3f-85fe-636041c73694" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevolvingCreditFacilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_019f905a-297e-7001-9e2c-9a40a9ed4433" xlink:to="loc_us-gaap_RevolvingCreditFacilityMember_019f905a-297e-7f3f-85fe-636041c73694" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommercialPaperMember_019f905a-297e-7f2a-9ca0-c4c0f7c68ea0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommercialPaperMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_019f905a-297e-7001-9e2c-9a40a9ed4433" xlink:to="loc_us-gaap_CommercialPaperMember_019f905a-297e-7f2a-9ca0-c4c0f7c68ea0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DerivativeNumberOfFairValueHedges_019f905a-297e-7f73-a39c-4825d9c8afe4" xlink:href="csx-20260930.xsd#csx_DerivativeNumberOfFairValueHedges"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_csx_DerivativeNumberOfFairValueHedges_019f905a-297e-7f73-a39c-4825d9c8afe4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeTermOfContract_019f905a-297e-7e02-814c-cb81e89d75a8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeTermOfContract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_DerivativeTermOfContract_019f905a-297e-7e02-814c-cb81e89d75a8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_019f905a-297e-789b-8696-cec71cac1b04" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_019f905a-297e-789b-8696-cec71cac1b04" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue_019f905a-297e-7789-a3e0-585c4e8304f0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue_019f905a-297e-7789-a3e0-585c4e8304f0" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestRateFairValueHedgeAssetAtFairValue_019f905a-297e-75b3-bc1c-c880b0571f51" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestRateFairValueHedgeAssetAtFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_InterestRateFairValueHedgeAssetAtFairValue_019f905a-297e-75b3-bc1c-c880b0571f51" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeAssetNotionalAmount_019f905a-297e-7fb3-ae00-8fe1ca9d67a7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeAssetNotionalAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_DerivativeAssetNotionalAmount_019f905a-297e-7fb3-ae00-8fe1ca9d67a7" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentInterestRateStatedPercentage_019f905a-297e-7a61-8e10-503f32731416" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_DebtInstrumentInterestRateStatedPercentage_019f905a-297e-7a61-8e10-503f32731416" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DerivativeInstrumentPeriodFollowingDebtMaturityHedgedTransaction_019f905a-297e-704b-96ce-06c9b447c621" xlink:href="csx-20260930.xsd#csx_DerivativeInstrumentPeriodFollowingDebtMaturityHedgedTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_csx_DerivativeInstrumentPeriodFollowingDebtMaturityHedgedTransaction_019f905a-297e-704b-96ce-06c9b447c621" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_DerivativeAssetUnsettledNotionalAmount_019f905a-297e-768f-971a-1a9680de9b2a" xlink:href="csx-20260930.xsd#csx_DerivativeAssetUnsettledNotionalAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_csx_DerivativeAssetUnsettledNotionalAmount_019f905a-297e-768f-971a-1a9680de9b2a" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_019f905a-297e-73e5-9b5c-42609ff058bf" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_019f905a-297e-73e5-9b5c-42609ff058bf" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCredit_019f905a-297e-78c8-a1dd-39267031ad68" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LineOfCredit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_019f905a-297e-73f1-8cb1-4c8ad9889829" xlink:to="loc_us-gaap_LineOfCredit_019f905a-297e-78c8-a1dd-39267031ad68" xlink:type="arc" order="10"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsScheduleofInterestRateDerivativesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLineItems_019f905a-297e-7769-9b2c-cf12e1240bda" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_019f905a-297e-7f27-b428-7ab583b18b4a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7769-9b2c-cf12e1240bda" xlink:to="loc_us-gaap_DebtInstrumentTable_019f905a-297e-7f27-b428-7ab583b18b4a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-71b7-b57f-5f56fd9a899f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019f905a-297e-7f27-b428-7ab583b18b4a" xlink:to="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-71b7-b57f-5f56fd9a899f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-71b7-b57f-5f56fd9a899f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-71b7-b57f-5f56fd9a899f" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-71b7-b57f-5f56fd9a899f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-70c6-9617-c4121fb70f6e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_019f905a-297e-71b7-b57f-5f56fd9a899f" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-70c6-9617-c4121fb70f6e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedRateNotesDueBetween2036And2040Member_019f905a-297e-7343-9a7d-be8470954fff" xlink:href="csx-20260930.xsd#csx_FixedRateNotesDueBetween2036And2040Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_019f905a-297e-70c6-9617-c4121fb70f6e" xlink:to="loc_csx_FixedRateNotesDueBetween2036And2040Member_019f905a-297e-7343-9a7d-be8470954fff" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e0b-a7d6-cbdb2906a546" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019f905a-297e-7f27-b428-7ab583b18b4a" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e0b-a7d6-cbdb2906a546" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7e0b-a7d6-cbdb2906a546_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e0b-a7d6-cbdb2906a546" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7e0b-a7d6-cbdb2906a546_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-797a-84a7-57886a0f41f0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e0b-a7d6-cbdb2906a546" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-797a-84a7-57886a0f41f0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FixedToFloatingInterestRateSwapMember_019f905a-297e-7c73-a5d9-0fe5a0db4fdb" xlink:href="csx-20260930.xsd#csx_FixedToFloatingInterestRateSwapMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-797a-84a7-57886a0f41f0" xlink:to="loc_csx_FixedToFloatingInterestRateSwapMember_019f905a-297e-7c73-a5d9-0fe5a0db4fdb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-7e83-9bf9-1a7d0addcc89" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019f905a-297e-7f27-b428-7ab583b18b4a" xlink:to="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-7e83-9bf9-1a7d0addcc89" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-7e83-9bf9-1a7d0addcc89_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-7e83-9bf9-1a7d0addcc89" xlink:to="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-7e83-9bf9-1a7d0addcc89_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-7bdd-9c13-6dc2086d1203" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-7e83-9bf9-1a7d0addcc89" xlink:to="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-7bdd-9c13-6dc2086d1203" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueHedgingMember_019f905a-297e-71a6-818f-7965873e3ce2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-7bdd-9c13-6dc2086d1203" xlink:to="loc_us-gaap_FairValueHedgingMember_019f905a-297e-71a6-818f-7965873e3ce2" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-7f4c-82dc-b6de9936b8be" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_019f905a-297e-7f27-b428-7ab583b18b4a" xlink:to="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-7f4c-82dc-b6de9936b8be" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-7f4c-82dc-b6de9936b8be_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-7f4c-82dc-b6de9936b8be" xlink:to="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-7f4c-82dc-b6de9936b8be_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-763e-9065-e11e4e0aca3c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-7f4c-82dc-b6de9936b8be" xlink:to="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-763e-9065-e11e4e0aca3c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DesignatedAsHedgingInstrumentMember_019f905a-297e-7c55-9722-6d776fdc1598" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DesignatedAsHedgingInstrumentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-763e-9065-e11e4e0aca3c" xlink:to="loc_us-gaap_DesignatedAsHedgingInstrumentMember_019f905a-297e-7c55-9722-6d776fdc1598" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_019f905a-297e-7d35-a629-b15be0a43ece" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7769-9b2c-cf12e1240bda" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_019f905a-297e-7d35-a629-b15be0a43ece" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgedAssetFairValueHedgeCumulativeIncreaseDecrease_019f905a-297e-7398-88a5-933a4542a41f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgedAssetFairValueHedgeCumulativeIncreaseDecrease"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7769-9b2c-cf12e1240bda" xlink:to="loc_us-gaap_HedgedAssetFairValueHedgeCumulativeIncreaseDecrease_019f905a-297e-7398-88a5-933a4542a41f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgedLiabilityFairValueHedgeCumulativeIncreaseDecrease_019f905a-297e-74b2-b0a8-c63b2f659f11" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgedLiabilityFairValueHedgeCumulativeIncreaseDecrease"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7769-9b2c-cf12e1240bda" xlink:to="loc_us-gaap_HedgedLiabilityFairValueHedgeCumulativeIncreaseDecrease_019f905a-297e-74b2-b0a8-c63b2f659f11" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgedLiabilityFairValueHedge_019f905a-297e-7c15-98ee-9ff79ceed772" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgedLiabilityFairValueHedge"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7769-9b2c-cf12e1240bda" xlink:to="loc_us-gaap_HedgedLiabilityFairValueHedge_019f905a-297e-7c15-98ee-9ff79ceed772" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/DebtandCreditAgreementsScheduleofInterestExpenseDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#DebtandCreditAgreementsScheduleofInterestExpenseDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/DebtandCreditAgreementsScheduleofInterestExpenseDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLineItems_019f905a-297e-7e28-9637-1fab13c369ea" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeTable_019f905a-297e-73df-93a3-a8c5dd755f70" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7e28-9637-1fab13c369ea" xlink:to="loc_us-gaap_DerivativeTable_019f905a-297e-73df-93a3-a8c5dd755f70" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7291-b25a-0ad880988b09" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DerivativeTable_019f905a-297e-73df-93a3-a8c5dd755f70" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7291-b25a-0ad880988b09" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7291-b25a-0ad880988b09_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7291-b25a-0ad880988b09" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7291-b25a-0ad880988b09_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7caa-b416-3a49163dea9d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7291-b25a-0ad880988b09" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7caa-b416-3a49163dea9d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FiveSeparateFixedToFloatingInterestRateSwapsMember_019f905a-297e-7d24-9690-bb196118934d" xlink:href="csx-20260930.xsd#csx_FiveSeparateFixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7caa-b416-3a49163dea9d" xlink:to="loc_csx_FiveSeparateFixedToFloatingInterestRateSwapsMember_019f905a-297e-7d24-9690-bb196118934d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestExpenseInterestBearingLiabilityIncreaseDecrease_019f905a-297e-778d-9cdf-3925371d6a20" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestExpenseInterestBearingLiabilityIncreaseDecrease"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeLineItems_019f905a-297e-7e28-9637-1fab13c369ea" xlink:to="loc_us-gaap_InterestExpenseInterestBearingLiabilityIncreaseDecrease_019f905a-297e-778d-9cdf-3925371d6a20" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/RevenuesScheduleofRevenuesDisaggregatedbyLinesofBusinessDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#RevenuesScheduleofRevenuesDisaggregatedbyLinesofBusinessDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/RevenuesScheduleofRevenuesDisaggregatedbyLinesofBusinessDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_019f905a-297e-7f9e-b8be-6bcc7dbc589c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_019f905a-297e-72ce-97a8-13826ff0ad21" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_019f905a-297e-7f9e-b8be-6bcc7dbc589c" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_019f905a-297e-72ce-97a8-13826ff0ad21" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_019f905a-297e-71d3-a4de-7e0eaaca0fae" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_019f905a-297e-72ce-97a8-13826ff0ad21" xlink:to="loc_srt_ProductOrServiceAxis_019f905a-297e-71d3-a4de-7e0eaaca0fae" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_019f905a-297e-71d3-a4de-7e0eaaca0fae_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_019f905a-297e-71d3-a4de-7e0eaaca0fae" xlink:to="loc_srt_ProductsAndServicesDomain_019f905a-297e-71d3-a4de-7e0eaaca0fae_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_019f905a-297e-71d3-a4de-7e0eaaca0fae" xlink:to="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:href="csx-20260930.xsd#csx_TotalMerchandiseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:to="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ChemicalsMember_019f905a-297e-70ac-b632-00343ba86a38" xlink:href="csx-20260930.xsd#csx_ChemicalsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_ChemicalsMember_019f905a-297e-70ac-b632-00343ba86a38" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_AgriculturalandFoodProductsMember_019f905a-297e-702c-bb3e-11e070d1d274" xlink:href="csx-20260930.xsd#csx_AgriculturalandFoodProductsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_AgriculturalandFoodProductsMember_019f905a-297e-702c-bb3e-11e070d1d274" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_AutomotiveMember_019f905a-297e-7b48-bb02-b19022691cfd" xlink:href="csx-20260930.xsd#csx_AutomotiveMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_AutomotiveMember_019f905a-297e-7b48-bb02-b19022691cfd" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_ForestProductsMember_019f905a-297e-7001-b70d-52c5131a0b66" xlink:href="csx-20260930.xsd#csx_ForestProductsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_ForestProductsMember_019f905a-297e-7001-b70d-52c5131a0b66" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_MetalsandEquipmentMember_019f905a-297e-7d1d-9a7e-60ca8d8e1449" xlink:href="csx-20260930.xsd#csx_MetalsandEquipmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_MetalsandEquipmentMember_019f905a-297e-7d1d-9a7e-60ca8d8e1449" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_MineralsMember_019f905a-297e-7096-a9ec-4f1dd5818412" xlink:href="csx-20260930.xsd#csx_MineralsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_MineralsMember_019f905a-297e-7096-a9ec-4f1dd5818412" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FertilizersMember_019f905a-297e-70c8-bf2e-90b8ca24e457" xlink:href="csx-20260930.xsd#csx_FertilizersMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_TotalMerchandiseMember_019f905a-297e-7e48-a1b7-92c1de9ae277" xlink:to="loc_csx_FertilizersMember_019f905a-297e-70c8-bf2e-90b8ca24e457" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_IntermodalMember_019f905a-297e-7925-8ea2-c7f12e60aafe" xlink:href="csx-20260930.xsd#csx_IntermodalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:to="loc_csx_IntermodalMember_019f905a-297e-7925-8ea2-c7f12e60aafe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_CoalServicesMember_019f905a-297e-7f49-863e-ba537a6d0dba" xlink:href="csx-20260930.xsd#csx_CoalServicesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:to="loc_csx_CoalServicesMember_019f905a-297e-7f49-863e-ba537a6d0dba" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TruckingMember_019f905a-297e-7835-a0d5-81ec4d1e59f0" xlink:href="csx-20260930.xsd#csx_TruckingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:to="loc_csx_TruckingMember_019f905a-297e-7835-a0d5-81ec4d1e59f0" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_OtherServicesMember_019f905a-297e-777f-bf5e-a90f21a2454d" xlink:href="csx-20260930.xsd#csx_OtherServicesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_019f905a-297e-7286-afd5-b91f87a7ddc9" xlink:to="loc_csx_OtherServicesMember_019f905a-297e-777f-bf5e-a90f21a2454d" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019f905a-297e-746e-bca5-bddc94541c9a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_019f905a-297e-7f9e-b8be-6bcc7dbc589c" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019f905a-297e-746e-bca5-bddc94541c9a" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/RevenuesScheduleofAccountsReceivableNetDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#RevenuesScheduleofAccountsReceivableNetDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/RevenuesScheduleofAccountsReceivableNetDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_019f905a-297e-77e8-b9e7-e897fedc422b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsNotesAndLoansReceivableLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable_019f905a-297e-79ac-8f7a-23559f6ec34c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_019f905a-297e-77e8-b9e7-e897fedc422b" xlink:to="loc_us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable_019f905a-297e-79ac-8f7a-23559f6ec34c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_019f905a-297e-74ef-b068-b9be65978e97" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAccountsNotesLoansAndFinancingReceivableTable_019f905a-297e-79ac-8f7a-23559f6ec34c" xlink:to="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_019f905a-297e-74ef-b068-b9be65978e97" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReceivableTypeDomain_019f905a-297e-74ef-b068-b9be65978e97_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ReceivableTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_019f905a-297e-74ef-b068-b9be65978e97" xlink:to="loc_us-gaap_ReceivableTypeDomain_019f905a-297e-74ef-b068-b9be65978e97_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReceivableTypeDomain_019f905a-297e-7747-a588-de1c083a495a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ReceivableTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AccountsNotesLoansAndFinancingReceivableByReceivableTypeAxis_019f905a-297e-74ef-b068-b9be65978e97" xlink:to="loc_us-gaap_ReceivableTypeDomain_019f905a-297e-7747-a588-de1c083a495a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FreightReceivablesMember_019f905a-297e-7c1a-b8af-f8937adda92e" xlink:href="csx-20260930.xsd#csx_FreightReceivablesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReceivableTypeDomain_019f905a-297e-7747-a588-de1c083a495a" xlink:to="loc_csx_FreightReceivablesMember_019f905a-297e-7c1a-b8af-f8937adda92e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NonFreightReceivablesMember_019f905a-297e-797c-9476-98a928a3367d" xlink:href="csx-20260930.xsd#csx_NonFreightReceivablesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReceivableTypeDomain_019f905a-297e-7747-a588-de1c083a495a" xlink:to="loc_csx_NonFreightReceivablesMember_019f905a-297e-797c-9476-98a928a3367d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsReceivableGross_019f905a-297e-7588-921f-9424864cd68c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsReceivableGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_019f905a-297e-77e8-b9e7-e897fedc422b" xlink:to="loc_us-gaap_AccountsReceivableGross_019f905a-297e-7588-921f-9424864cd68c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllowanceForDoubtfulAccountsReceivable_019f905a-297e-7fe6-8d33-fe3d7b68e6bf" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllowanceForDoubtfulAccountsReceivable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_019f905a-297e-77e8-b9e7-e897fedc422b" xlink:to="loc_us-gaap_AllowanceForDoubtfulAccountsReceivable_019f905a-297e-7fe6-8d33-fe3d7b68e6bf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccountsReceivableNet_019f905a-297e-708b-8df3-3ecb07bf970f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsReceivableNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccountsNotesAndLoansReceivableLineItems_019f905a-297e-77e8-b9e7-e897fedc422b" xlink:to="loc_us-gaap_AccountsReceivableNet_019f905a-297e-708b-8df3-3ecb07bf970f" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsScheduleofFairValueofInvestmentAssetsDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#FairValueMeasurementsScheduleofFairValueofInvestmentAssetsDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsScheduleofFairValueofInvestmentAssetsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-76d7-a134-bfa2ba54c00c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7267-aba4-fc455fdf9383" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-76d7-a134-bfa2ba54c00c" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7267-aba4-fc455fdf9383" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-71b1-9c4c-3bf4a3e55984" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7267-aba4-fc455fdf9383" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-71b1-9c4c-3bf4a3e55984" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-71b1-9c4c-3bf4a3e55984_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-71b1-9c4c-3bf4a3e55984" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-71b1-9c4c-3bf4a3e55984_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7bf7-a527-b1ff3701f3cb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-71b1-9c4c-3bf4a3e55984" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7bf7-a527-b1ff3701f3cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_019f905a-297e-7cf4-84ff-940d40402fcb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7bf7-a527-b1ff3701f3cb" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_019f905a-297e-7cf4-84ff-940d40402fcb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_019f905a-297e-79dc-9638-f888b0725cb4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7bf7-a527-b1ff3701f3cb" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_019f905a-297e-79dc-9638-f888b0725cb4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FairValueInputsLevel1AndLevel2Member_019f905a-297e-793a-b383-6106c97c399d" xlink:href="csx-20260930.xsd#csx_FairValueInputsLevel1AndLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7bf7-a527-b1ff3701f3cb" xlink:to="loc_csx_FairValueInputsLevel1AndLevel2Member_019f905a-297e-793a-b383-6106c97c399d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_019f905a-297e-7996-84d4-e9fbc53ec59c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7267-aba4-fc455fdf9383" xlink:to="loc_us-gaap_FinancialInstrumentAxis_019f905a-297e-7996-84d4-e9fbc53ec59c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-7996-84d4-e9fbc53ec59c_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_019f905a-297e-7996-84d4-e9fbc53ec59c" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-7996-84d4-e9fbc53ec59c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_019f905a-297e-7996-84d4-e9fbc53ec59c" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ExchangeTradedFundsMember_019f905a-297e-7678-86b1-4e9ff2633a0a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ExchangeTradedFundsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:to="loc_us-gaap_ExchangeTradedFundsMember_019f905a-297e-7678-86b1-4e9ff2633a0a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BankTimeDepositsMember_019f905a-297e-7f93-8d33-0a373a4928f0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BankTimeDepositsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:to="loc_us-gaap_BankTimeDepositsMember_019f905a-297e-7f93-8d33-0a373a4928f0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryAndGovernmentMember_019f905a-297e-7b40-b362-113e63e60b9c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USTreasuryAndGovernmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:to="loc_us-gaap_USTreasuryAndGovernmentMember_019f905a-297e-7b40-b362-113e63e60b9c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CorporateBondSecuritiesMember_019f905a-297e-77b8-8460-f0600923997e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CorporateBondSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:to="loc_us-gaap_CorporateBondSecuritiesMember_019f905a-297e-77b8-8460-f0600923997e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetBackedSecuritiesMember_019f905a-297e-7aa1-870e-87f3c0dd4cc4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetBackedSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_019f905a-297e-75fe-84fa-06e2af864b1e" xlink:to="loc_us-gaap_AssetBackedSecuritiesMember_019f905a-297e-7aa1-870e-87f3c0dd4cc4" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentsFairValueDisclosure_019f905a-297e-7ba9-8b1a-680c63ed53ea" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-76d7-a134-bfa2ba54c00c" xlink:to="loc_us-gaap_InvestmentsFairValueDisclosure_019f905a-297e-7ba9-8b1a-680c63ed53ea" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsNarrativeDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#FairValueMeasurementsNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_019f905a-297e-78f5-bbc8-b78c79caf02d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_019f905a-297e-71cf-a83d-168bb3781338" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_019f905a-297e-78f5-bbc8-b78c79caf02d" xlink:to="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_019f905a-297e-71cf-a83d-168bb3781338" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-764a-9ad7-26f33f3041cb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByMeasurementBasisAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_019f905a-297e-71cf-a83d-168bb3781338" xlink:to="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-764a-9ad7-26f33f3041cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-764a-9ad7-26f33f3041cb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-764a-9ad7-26f33f3041cb" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-764a-9ad7-26f33f3041cb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7484-a28d-4185a54c2656" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueDisclosureItemAmountsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-764a-9ad7-26f33f3041cb" xlink:to="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7484-a28d-4185a54c2656" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-725e-b23d-c83a1bda2dff" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7484-a28d-4185a54c2656" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-725e-b23d-c83a1bda2dff" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_019f905a-297e-7eed-add2-12e30c2a8ae8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EstimateOfFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-725e-b23d-c83a1bda2dff" xlink:to="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_019f905a-297e-7eed-add2-12e30c2a8ae8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_019f905a-297e-75fb-ac82-66456081bfd2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CarryingReportedAmountFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-725e-b23d-c83a1bda2dff" xlink:to="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_019f905a-297e-75fb-ac82-66456081bfd2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e44-881c-72a332a812eb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentRiskAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_019f905a-297e-71cf-a83d-168bb3781338" xlink:to="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e44-881c-72a332a812eb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7e44-881c-72a332a812eb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e44-881c-72a332a812eb" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-7e44-881c-72a332a812eb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-73b4-80b6-52108de74102" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentRiskAxis_019f905a-297e-7e44-881c-72a332a812eb" xlink:to="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-73b4-80b6-52108de74102" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_A20232025FixedToFloatingInterestRateSwapsMember_019f905a-297e-73ca-8913-e348c56075e6" xlink:href="csx-20260930.xsd#csx_A20232025FixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-73b4-80b6-52108de74102" xlink:to="loc_csx_A20232025FixedToFloatingInterestRateSwapsMember_019f905a-297e-73ca-8913-e348c56075e6" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_A2022FixedToFloatingInterestRateSwapsMember_019f905a-297e-7114-abea-709d1b42fc51" xlink:href="csx-20260930.xsd#csx_A2022FixedToFloatingInterestRateSwapsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DerivativeContractTypeDomain_019f905a-297e-73b4-80b6-52108de74102" xlink:to="loc_csx_A2022FixedToFloatingInterestRateSwapsMember_019f905a-297e-7114-abea-709d1b42fc51" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-743b-9f67-f865985a6655" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_019f905a-297e-71cf-a83d-168bb3781338" xlink:to="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-743b-9f67-f865985a6655" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-743b-9f67-f865985a6655_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-743b-9f67-f865985a6655" xlink:to="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-743b-9f67-f865985a6655_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-70ae-9a04-4b428183240f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingRelationshipDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DerivativeInstrumentsGainLossByHedgingRelationshipAxis_019f905a-297e-743b-9f67-f865985a6655" xlink:to="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-70ae-9a04-4b428183240f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueHedgingMember_019f905a-297e-73cd-97c5-5f763f6a19bd" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueHedgingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingRelationshipDomain_019f905a-297e-70ae-9a04-4b428183240f" xlink:to="loc_us-gaap_FairValueHedgingMember_019f905a-297e-73cd-97c5-5f763f6a19bd" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-74bf-9571-339878ddff08" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_019f905a-297e-71cf-a83d-168bb3781338" xlink:to="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-74bf-9571-339878ddff08" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-74bf-9571-339878ddff08_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-74bf-9571-339878ddff08" xlink:to="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-74bf-9571-339878ddff08_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-70b0-94bd-6f9696d005d9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_HedgingDesignationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_HedgingDesignationAxis_019f905a-297e-74bf-9571-339878ddff08" xlink:to="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-70b0-94bd-6f9696d005d9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DesignatedAsHedgingInstrumentMember_019f905a-297e-7ee9-b134-d29d16fd04b5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DesignatedAsHedgingInstrumentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_HedgingDesignationDomain_019f905a-297e-70b0-94bd-6f9696d005d9" xlink:to="loc_us-gaap_DesignatedAsHedgingInstrumentMember_019f905a-297e-7ee9-b134-d29d16fd04b5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_019f905a-297e-77be-ae70-3223f3661751" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_019f905a-297e-78f5-bbc8-b78c79caf02d" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_019f905a-297e-77be-ae70-3223f3661751" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_019f905a-297e-7228-b766-495f628a67cd" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_019f905a-297e-78f5-bbc8-b78c79caf02d" xlink:to="loc_us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis_019f905a-297e-7228-b766-495f628a67cd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue_019f905a-297e-7fa4-8ff2-88f67b4f78d7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_019f905a-297e-78f5-bbc8-b78c79caf02d" xlink:to="loc_us-gaap_InterestRateFairValueHedgeLiabilityAtFairValue_019f905a-297e-7fa4-8ff2-88f67b4f78d7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestRateFairValueHedgeAssetAtFairValue_019f905a-297e-760d-a7af-b04ebecdbae3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestRateFairValueHedgeAssetAtFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_019f905a-297e-78f5-bbc8-b78c79caf02d" xlink:to="loc_us-gaap_InterestRateFairValueHedgeAssetAtFairValue_019f905a-297e-760d-a7af-b04ebecdbae3" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsScheduleofInvestmentMaturitiesDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#FairValueMeasurementsScheduleofInvestmentMaturitiesDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsScheduleofInvestmentMaturitiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7268-a228-f22fd10c8ac7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7268-a228-f22fd10c8ac7" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d50-a17c-bdb3b72d2101" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByMeasurementBasisAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7268-a228-f22fd10c8ac7" xlink:to="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d50-a17c-bdb3b72d2101" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7d50-a17c-bdb3b72d2101_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d50-a17c-bdb3b72d2101" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7d50-a17c-bdb3b72d2101_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7b58-8ac8-3d8f264ad419" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueDisclosureItemAmountsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d50-a17c-bdb3b72d2101" xlink:to="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7b58-8ac8-3d8f264ad419" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7bc6-9f46-33d3c7d1594d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7b58-8ac8-3d8f264ad419" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7bc6-9f46-33d3c7d1594d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_019f905a-297e-7595-8fd5-e82c72ab6932" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EstimateOfFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7bc6-9f46-33d3c7d1594d" xlink:to="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_019f905a-297e-7595-8fd5-e82c72ab6932" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_InvestmentsMaturityAllocatedAndSingleMaturityDateRollingWithinOneYearFairValue_019f905a-297e-79db-9d4d-47d5e748f457" xlink:href="csx-20260930.xsd#csx_InvestmentsMaturityAllocatedAndSingleMaturityDateRollingWithinOneYearFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:to="loc_csx_InvestmentsMaturityAllocatedAndSingleMaturityDateRollingWithinOneYearFairValue_019f905a-297e-79db-9d4d-47d5e748f457" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_InvestmentsFairValueMaturityAllocatedAndSingleMaturityDateAfterYearOneThroughFive_019f905a-297e-7012-b0b5-aed05c67b038" xlink:href="csx-20260930.xsd#csx_InvestmentsFairValueMaturityAllocatedAndSingleMaturityDateAfterYearOneThroughFive"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:to="loc_csx_InvestmentsFairValueMaturityAllocatedAndSingleMaturityDateAfterYearOneThroughFive_019f905a-297e-7012-b0b5-aed05c67b038" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_InvestmentsFairValueMaturityAllocatedAndSingleMaturityDateAfterYear5Through10_019f905a-297e-78c0-aa0f-a1c18ba26ad0" xlink:href="csx-20260930.xsd#csx_InvestmentsFairValueMaturityAllocatedAndSingleMaturityDateAfterYear5Through10"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:to="loc_csx_InvestmentsFairValueMaturityAllocatedAndSingleMaturityDateAfterYear5Through10_019f905a-297e-78c0-aa0f-a1c18ba26ad0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_InvestmentsMaturityAllocatedAndSingleMaturityDateRollingAfter10YearsFairValue_019f905a-297e-749e-84de-e61615fd5f54" xlink:href="csx-20260930.xsd#csx_InvestmentsMaturityAllocatedAndSingleMaturityDateRollingAfter10YearsFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:to="loc_csx_InvestmentsMaturityAllocatedAndSingleMaturityDateRollingAfter10YearsFairValue_019f905a-297e-749e-84de-e61615fd5f54" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InvestmentsFairValueDisclosure_019f905a-297e-7274-b28f-379804591f1e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-77b2-a7a9-f52272263614" xlink:to="loc_us-gaap_InvestmentsFairValueDisclosure_019f905a-297e-7274-b28f-379804591f1e" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/FairValueMeasurementsScheduleofFairValueandCarryingValueofLongTermDebtDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#FairValueMeasurementsScheduleofFairValueandCarryingValueofLongTermDebtDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/FairValueMeasurementsScheduleofFairValueandCarryingValueofLongTermDebtDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-7ec8-894e-d107707f8a09" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7629-a32b-2db5516415b3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByBalanceSheetGroupingTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-7ec8-894e-d107707f8a09" xlink:to="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7629-a32b-2db5516415b3" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-7248-9408-c5e0109b66e1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7629-a32b-2db5516415b3" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-7248-9408-c5e0109b66e1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7248-9408-c5e0109b66e1_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-7248-9408-c5e0109b66e1" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-7248-9408-c5e0109b66e1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-72ea-a348-017e485986b1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_019f905a-297e-7248-9408-c5e0109b66e1" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-72ea-a348-017e485986b1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_019f905a-297e-74f0-90dc-81394f062f15" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_019f905a-297e-72ea-a348-017e485986b1" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_019f905a-297e-74f0-90dc-81394f062f15" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d33-b734-83ed8f268ee6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByMeasurementBasisAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueByBalanceSheetGroupingTable_019f905a-297e-7629-a32b-2db5516415b3" xlink:to="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d33-b734-83ed8f268ee6" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7d33-b734-83ed8f268ee6_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d33-b734-83ed8f268ee6" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7d33-b734-83ed8f268ee6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7935-bfb2-91683daca93f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueDisclosureItemAmountsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementBasisAxis_019f905a-297e-7d33-b734-83ed8f268ee6" xlink:to="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7935-bfb2-91683daca93f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7377-9920-1b26fab52cb0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PortionAtFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureItemAmountsDomain_019f905a-297e-7935-bfb2-91683daca93f" xlink:to="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7377-9920-1b26fab52cb0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_019f905a-297e-7b55-8a0b-6732ba113e7e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EstimateOfFairValueFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7377-9920-1b26fab52cb0" xlink:to="loc_us-gaap_EstimateOfFairValueFairValueDisclosureMember_019f905a-297e-7b55-8a0b-6732ba113e7e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_019f905a-297e-77c0-b880-66bfde70167f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CarryingReportedAmountFairValueDisclosureMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PortionAtFairValueFairValueDisclosureMember_019f905a-297e-7377-9920-1b26fab52cb0" xlink:to="loc_us-gaap_CarryingReportedAmountFairValueDisclosureMember_019f905a-297e-77c0-b880-66bfde70167f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFairValue_019f905a-297e-732b-8813-4edc57f99497" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueBalanceSheetGroupingFinancialStatementCaptionsLineItems_019f905a-297e-7ec8-894e-d107707f8a09" xlink:to="loc_us-gaap_DebtInstrumentFairValue_019f905a-297e-732b-8813-4edc57f99497" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/OtherComprehensiveIncomeLossScheduleofChangesintheAOCIBalancebyComponentDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#OtherComprehensiveIncomeLossScheduleofChangesintheAOCIBalancebyComponentDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/OtherComprehensiveIncomeLossScheduleofChangesintheAOCIBalancebyComponentDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems_019f905a-297e-7d22-a6cf-0fca781059e8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossTable_019f905a-297e-75ca-abbe-78007b336ae0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeLossTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems_019f905a-297e-7d22-a6cf-0fca781059e8" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossTable_019f905a-297e-75ca-abbe-78007b336ae0" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297e-7fb1-a6d0-0bf1ade0a7e4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossTable_019f905a-297e-75ca-abbe-78007b336ae0" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297e-7fb1-a6d0-0bf1ade0a7e4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019f905a-297e-7fb1-a6d0-0bf1ade0a7e4_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297e-7fb1-a6d0-0bf1ade0a7e4" xlink:to="loc_us-gaap_EquityComponentDomain_019f905a-297e-7fb1-a6d0-0bf1ade0a7e4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019f905a-297e-7a7b-8989-78a2e3010c43" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019f905a-297e-7fb1-a6d0-0bf1ade0a7e4" xlink:to="loc_us-gaap_EquityComponentDomain_019f905a-297e-7a7b-8989-78a2e3010c43" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297e-71bb-b2bf-471b7c016d8e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019f905a-297e-7a7b-8989-78a2e3010c43" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297e-71bb-b2bf-471b7c016d8e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember_019f905a-297e-760a-89e2-0ae68256d688" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297e-71bb-b2bf-471b7c016d8e" xlink:to="loc_us-gaap_AccumulatedDefinedBenefitPlansAdjustmentMember_019f905a-297e-760a-89e2-0ae68256d688" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember_019f905a-297e-7614-879e-49d2bc2e4983" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297e-71bb-b2bf-471b7c016d8e" xlink:to="loc_us-gaap_AccumulatedGainLossNetCashFlowHedgeParentMember_019f905a-297e-7614-879e-49d2bc2e4983" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_AccumulatedOtherAdjustmentstoParentMember_019f905a-297e-704a-989c-44daf49528e8" xlink:href="csx-20260930.xsd#csx_AccumulatedOtherAdjustmentstoParentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019f905a-297e-71bb-b2bf-471b7c016d8e" xlink:to="loc_csx_AccumulatedOtherAdjustmentstoParentMember_019f905a-297e-704a-989c-44daf49528e8" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_019f905a-297e-7b3f-9189-8db13b9eea82" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AccumulatedOtherComprehensiveIncomeLossLineItems_019f905a-297e-7d22-a6cf-0fca781059e8" xlink:to="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_019f905a-297e-7b3f-9189-8db13b9eea82" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_019f905a-297e-7651-807b-0e78064b5737" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_019f905a-297e-7b3f-9189-8db13b9eea82" xlink:to="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_019f905a-297e-7651-807b-0e78064b5737" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodChangeAbstract_019f905a-297e-738d-83c2-14dde80e6dde" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodChangeAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodNetOfTaxAbstract_019f905a-297e-7b3f-9189-8db13b9eea82" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodChangeAbstract_019f905a-297e-738d-83c2-14dde80e6dde" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodBeforeTax_019f905a-297e-7f21-ad9b-98c347d32f7b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodChangeAbstract_019f905a-297e-738d-83c2-14dde80e6dde" xlink:to="loc_us-gaap_ReclassificationFromAccumulatedOtherComprehensiveIncomeCurrentPeriodBeforeTax_019f905a-297e-7f21-ad9b-98c347d32f7b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReclassificationFromAociCurrentPeriodTax_019f905a-297e-760f-814b-b3a6f08b6b71" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ReclassificationFromAociCurrentPeriodTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodChangeAbstract_019f905a-297e-738d-83c2-14dde80e6dde" xlink:to="loc_us-gaap_ReclassificationFromAociCurrentPeriodTax_019f905a-297e-760f-814b-b3a6f08b6b71" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTax_019f905a-297e-763c-9d95-8bd455603977" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherComprehensiveIncomeLossNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTaxPeriodChangeAbstract_019f905a-297e-738d-83c2-14dde80e6dde" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossNetOfTax_019f905a-297e-763c-9d95-8bd455603977" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_019f905a-297e-7467-a3c7-6bdf3783dc21" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/SegmentReportingandSignificantExpensesNarrativeDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#SegmentReportingandSignificantExpensesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/SegmentReportingandSignificantExpensesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7388-b584-d34466f65493" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7862-955b-7e9e594b6cb4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7388-b584-d34466f65493" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7862-955b-7e9e594b6cb4" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7dca-8b4d-21caa94bb96b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7862-955b-7e9e594b6cb4" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7dca-8b4d-21caa94bb96b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019f905a-297e-7dca-8b4d-21caa94bb96b_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7dca-8b4d-21caa94bb96b" xlink:to="loc_us-gaap_SegmentDomain_019f905a-297e-7dca-8b4d-21caa94bb96b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019f905a-297e-740d-9fe8-376f95b5ed38" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7dca-8b4d-21caa94bb96b" xlink:to="loc_us-gaap_SegmentDomain_019f905a-297e-740d-9fe8-376f95b5ed38" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_RailSegmentMember_019f905a-297e-726d-bbdc-4ce81dc3f7f1" xlink:href="csx-20260930.xsd#csx_RailSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_019f905a-297e-740d-9fe8-376f95b5ed38" xlink:to="loc_csx_RailSegmentMember_019f905a-297e-726d-bbdc-4ce81dc3f7f1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NumberOfOperatingSegments_019f905a-297e-7628-8ca2-e99443d01d7b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NumberOfOperatingSegments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7388-b584-d34466f65493" xlink:to="loc_us-gaap_NumberOfOperatingSegments_019f905a-297e-7628-8ca2-e99443d01d7b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_NumberOfReportableSegmentsNotDisclosedFlag_019f905a-297e-75a8-beb8-1442339f35fd" xlink:href="csx-20260930.xsd#csx_NumberOfReportableSegmentsNotDisclosedFlag"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7388-b584-d34466f65493" xlink:to="loc_csx_NumberOfReportableSegmentsNotDisclosedFlag_019f905a-297e-75a8-beb8-1442339f35fd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentExpenditureAdditionToLongLivedAssets_019f905a-297e-7ca6-b44b-28b2635f6789" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentExpenditureAdditionToLongLivedAssets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7388-b584-d34466f65493" xlink:to="loc_us-gaap_SegmentExpenditureAdditionToLongLivedAssets_019f905a-297e-7ca6-b44b-28b2635f6789" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_Assets_019f905a-297e-7480-9318-fce634aa3186" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Assets"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7388-b584-d34466f65493" xlink:to="loc_us-gaap_Assets_019f905a-297e-7480-9318-fce634aa3186" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.csx.com/role/SegmentReportingandSignificantExpensesScheduleofSegmentReportingInformationDetails" xlink:type="simple" xlink:href="csx-20260930.xsd#SegmentReportingandSignificantExpensesScheduleofSegmentReportingInformationDetails"/>
  <link:definitionLink xlink:role="http://www.csx.com/role/SegmentReportingandSignificantExpensesScheduleofSegmentReportingInformationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7112-8553-ac9d5b9c9e16" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7112-8553-ac9d5b9c9e16" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7406-98e3-5d9893a4cb6e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7112-8553-ac9d5b9c9e16" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7406-98e3-5d9893a4cb6e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019f905a-297e-7406-98e3-5d9893a4cb6e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7406-98e3-5d9893a4cb6e" xlink:to="loc_us-gaap_SegmentDomain_019f905a-297e-7406-98e3-5d9893a4cb6e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019f905a-297e-715a-ac75-1991e8f8d429" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019f905a-297e-7406-98e3-5d9893a4cb6e" xlink:to="loc_us-gaap_SegmentDomain_019f905a-297e-715a-ac75-1991e8f8d429" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_RailSegmentMember_019f905a-297e-7279-a1de-0b5d57a900aa" xlink:href="csx-20260930.xsd#csx_RailSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_019f905a-297e-715a-ac75-1991e8f8d429" xlink:to="loc_csx_RailSegmentMember_019f905a-297e-7279-a1de-0b5d57a900aa" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_TruckingSegmentMember_019f905a-297e-7342-80cd-64933122c82f" xlink:href="csx-20260930.xsd#csx_TruckingSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_019f905a-297e-715a-ac75-1991e8f8d429" xlink:to="loc_csx_TruckingSegmentMember_019f905a-297e-7342-80cd-64933122c82f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ConsolidationItemsAxis_019f905a-297e-7ae7-9af9-0b0cb3187ffc" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7112-8553-ac9d5b9c9e16" xlink:to="loc_srt_ConsolidationItemsAxis_019f905a-297e-7ae7-9af9-0b0cb3187ffc" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ConsolidationItemsDomain_019f905a-297e-7ae7-9af9-0b0cb3187ffc_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ConsolidationItemsAxis_019f905a-297e-7ae7-9af9-0b0cb3187ffc" xlink:to="loc_srt_ConsolidationItemsDomain_019f905a-297e-7ae7-9af9-0b0cb3187ffc_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ConsolidationItemsDomain_019f905a-297e-77dd-9180-821355d59fc1" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ConsolidationItemsAxis_019f905a-297e-7ae7-9af9-0b0cb3187ffc" xlink:to="loc_srt_ConsolidationItemsDomain_019f905a-297e-77dd-9180-821355d59fc1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingSegmentsMember_019f905a-297e-7613-ba47-835bb6d7b3fb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingSegmentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ConsolidationItemsDomain_019f905a-297e-77dd-9180-821355d59fc1" xlink:to="loc_us-gaap_OperatingSegmentsMember_019f905a-297e-7613-ba47-835bb6d7b3fb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IntersegmentEliminationMember_019f905a-297e-7ca9-92c9-31cccf35be80" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IntersegmentEliminationMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ConsolidationItemsDomain_019f905a-297e-77dd-9180-821355d59fc1" xlink:to="loc_us-gaap_IntersegmentEliminationMember_019f905a-297e-7ca9-92c9-31cccf35be80" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelTypeAxis_019f905a-297e-7cde-b69d-c9498297a486" xlink:href="csx-20260930.xsd#csx_FuelTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019f905a-297e-7112-8553-ac9d5b9c9e16" xlink:to="loc_csx_FuelTypeAxis_019f905a-297e-7cde-b69d-c9498297a486" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelTypeDomain_019f905a-297e-7cde-b69d-c9498297a486_default" xlink:href="csx-20260930.xsd#csx_FuelTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_csx_FuelTypeAxis_019f905a-297e-7cde-b69d-c9498297a486" xlink:to="loc_csx_FuelTypeDomain_019f905a-297e-7cde-b69d-c9498297a486_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelTypeDomain_019f905a-297e-78d0-8ec8-f19b53ad7057" xlink:href="csx-20260930.xsd#csx_FuelTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_csx_FuelTypeAxis_019f905a-297e-7cde-b69d-c9498297a486" xlink:to="loc_csx_FuelTypeDomain_019f905a-297e-78d0-8ec8-f19b53ad7057" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelLocomotiveMember_019f905a-297e-7b28-857d-f81e466b1a51" xlink:href="csx-20260930.xsd#csx_FuelLocomotiveMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_FuelTypeDomain_019f905a-297e-78d0-8ec8-f19b53ad7057" xlink:to="loc_csx_FuelLocomotiveMember_019f905a-297e-7b28-857d-f81e466b1a51" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_FuelNonLocomotiveMember_019f905a-297e-760a-939d-635fac4a1748" xlink:href="csx-20260930.xsd#csx_FuelNonLocomotiveMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_csx_FuelTypeDomain_019f905a-297e-78d0-8ec8-f19b53ad7057" xlink:to="loc_csx_FuelNonLocomotiveMember_019f905a-297e-760a-939d-635fac4a1748" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019f905a-297e-755c-a924-6f64e6e55104" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_019f905a-297e-755c-a924-6f64e6e55104" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LaborAndRelatedExpense_019f905a-297e-7db3-9f27-96b3dcc0ee11" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LaborAndRelatedExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_LaborAndRelatedExpense_019f905a-297e-7db3-9f27-96b3dcc0ee11" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherCostAndExpenseOperating_019f905a-297e-7780-9f27-6cece8a0fb5f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherCostAndExpenseOperating"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_OtherCostAndExpenseOperating_019f905a-297e-7780-9f27-6cece8a0fb5f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DepreciationDepletionAndAmortization_019f905a-297e-790c-9659-9fba372e7269" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DepreciationDepletionAndAmortization"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_DepreciationDepletionAndAmortization_019f905a-297e-790c-9659-9fba372e7269" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FuelCosts_019f905a-297e-785c-8adc-5d47203073c3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FuelCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_FuelCosts_019f905a-297e-785c-8adc-5d47203073c3" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_csx_EquipmentAndOtherRents_019f905a-297e-7984-8aef-e6e81550f9f9" xlink:href="csx-20260930.xsd#csx_EquipmentAndOtherRents"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_csx_EquipmentAndOtherRents_019f905a-297e-7984-8aef-e6e81550f9f9" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GainLossOnSaleOfPropertyPlantEquipment_019f905a-297e-7472-9aff-55bad4c5d3d3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GainLossOnSaleOfPropertyPlantEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_GainLossOnSaleOfPropertyPlantEquipment_019f905a-297e-7472-9aff-55bad4c5d3d3" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingIncomeLoss_019f905a-297e-7bdd-aac8-34b7dd441ef2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_OperatingIncomeLoss_019f905a-297e-7bdd-aac8-34b7dd441ef2" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingCostsAndExpenses_019f905a-297e-7034-88a9-4f6d6b9fbf9e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingCostsAndExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_OperatingCostsAndExpenses_019f905a-297e-7034-88a9-4f6d6b9fbf9e" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterestExpenseNonoperating_019f905a-297e-7349-bbdf-2436b47daf83" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterestExpenseNonoperating"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_InterestExpenseNonoperating_019f905a-297e-7349-bbdf-2436b47daf83" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NonoperatingIncomeExpense_019f905a-297e-7dca-9e74-db3bde424831" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_NonoperatingIncomeExpense_019f905a-297e-7dca-9e74-db3bde424831" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest_019f905a-297e-7584-9be8-3d8077252df0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest_019f905a-297e-7584-9be8-3d8077252df0" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GoodwillImpairmentLoss_01a01061-7814-7bfa-97e8-aca8b7ad37a5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GoodwillImpairmentLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019f905a-297e-7171-bac6-6d14c4be18cf" xlink:to="loc_us-gaap_GoodwillImpairmentLoss_01a01061-7814-7bfa-97e8-aca8b7ad37a5" xlink:type="arc" order="12"/>
  </link:definitionLink>
</link:linkbase>
