Chloroethylnitrosoureas　(CENUs)　are　important　chemotherapies　applied　in　the　treatment　of　cancer.　They　exert　anticancer　activity　by　inducing　DNA　interstrand　cross-links　(ICLs)　via　the　formation　of　two　O-6-alkylguanine　intermediates,　O-6-chloroethylguanine　(O-6-ClEtG)　and　N1,O-6-ethanoguanine　(N1,O-6-EtG).　However,　O-6-alkylguanine-DNA　alkyltransferase　(AGT),　a　DNA-repair　enzyme,　can　restore　the　O-6-alkylguanine　damages　and　thereby　obstruct　the　formation　of　ICLs　(dG-dC　cross-link).　In　this　study,　the　inhibitory　mechanism　of　ICL　formation　was　investigated　to　elucidate　the　drug　resistance　of　CENUs　mediated　by　AGT　in　detail.　Based　on　the　structures　of　the　substrate-enzyme　complexes　obtained　from　docking　and　MD　simulations,　two　ONIOM　(QM/MM)　models　with　different　sizes　of　the　QM　region　were　constructed.　The　model　with　a　larger　QM　region,　which　included　the　substrate　(O-6-ClEtG　or　N1,O-6-EtG),　a　water　molecule,　and　five　residues　(Tyr114,　Cys145,　His146,　Lys165,　and　Glu172)　in　the　active　pocket　of　AGT,　accurately　described　the　repairing　reaction　and　generated　the　results　coinciding　with　the　experimental　outcomes.　The　repair　process　consists　of　two　sequential　steps:　hydrogen　transfer　to　form　a　thiolate　anion　on　Cys145　and　alkyl　transfer　from　the　O-6　site　of　guanine　(the　rate-limiting　step).　The　repair　of　N1,O-6-EtG　was　more　favorable　than　that　of　O-6-ClEtG　from　both　kinetics　and　thermodynamics　aspects.　Moreover,　the　comparison　of　the　repairing　process　with　the　formation　of　dG-dC　cross-link　and　the　inhibition　of　AGT　by　O-6-benzylguanine　(O-6-BG)　showed　that　the　presence　of　AGT　could　effectively　interrupt　the　formation　of　ICLs　leading　to　drug　resistance,　and　the　inhibition　of　AGT　by　O-6-BG　that　was　energetically　more　favorable　than　the　repair　of　O-6-ClEtG　could　not　prevent　the　repair　of　N1,O-6-EtG.　Therefore,　it　is　necessary　to　completely　eliminate　AGT　activity　before　CENUs　medication　to　enhance　the　chemotherapeutic　effectiveness.　This　work　provides　reasonable　explanations　for　the　supposed　mechanism　of　AGT-mediated　drug　resistance　of　CENUs　and　will　assist　in　the　development　of　novel　CENU　chemotherapies　and　their　medication　strategies.