Chloroethylnitmsoureas　(CENUs)　are　an　important　family　of　chemotherapies　in　clinical　treatment　of　cancers,　which　exert　antitumor　activity　by　inducing　the　formation　of　DNA　interstrand　crosslinks　(dG-dC　ICLs).　However,　the　drug　resistance　mediated　by　O-6-alkylguanine-DNA　alkyltransferase　(AGT)　and　absence　of　tumor-targeting　ability　largely　decrease　the　antitumor　efficacy　of　CENUs.　In　this　study,　we　synthesized　an　azobenzene-based　hypoxia-activated　combi-nitrosourea　prodrug,　AzoBGNU,　and　evaluated　its　hypoxic　selectivity　and　antitumor　activity.　The　prodrug　was　composed　of　a　CENU　pharmacophore　and　an　O-6-benzylguanine　(O-6-BG)　analog　moiety　masked　by　a　N,N-dimethyl-4-(phenyldiazenyl)aniline　segment　as　a　hypoxia-activated　trigger,　which　was　designed　to　be　selectively　reduced　via　azo　bond　break　in　hypoxic　tumor　microenvironment,　accompanied　with　releasing　of　an　O-6-BG　analog　to　inhibit　AGT　and　a　chloroethylating　agent　to　induce　dG-dC　ICLs.　AzoBGNU　exhibited　significantly　increased　cytotoxicity　and　apoptosis-inducing　ability　toward　DU145　cells　under　hypoxia　compared　with　normoxia,　indicating　the　hypoxia-responsiveness　as　expected.　Predominant　higher　cytotoxicity　was　observed　in　the　cells　treated　by　AzoBGNU　than　those　by　traditional　CENU　chemotherapy　ACNU　and　its　combination　with　O-6--BG.　The　levels　of　dG-dC　ICLs　in　DU145　cells　induced　by　AzoBGNU　was　remarkably　enhanced　under　hypoxia,　which　was　approximately　6-fold　higher　than　those　in　the　AzoBGNU-treated　groups　under　normoxia　and　those　in　the　ACNU-treated　groups.　The　results　demonstrated　that　azobenzene-based　combinitrosourea　prodrug　possessed　desirable　tumor-hypoxia　targeting　ability　and　eliminated　chemoresistance　compared　with　the　conventional　CENUs.