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Background Alth6ugh norcanthnridin (NCTD) has been used to treat tumors in China for many years, its metabolism pathway and the relevant enzymes still remain unclear now.The primary biotransformation product of norcantharidin in vivo is norcantharidin diacid because of the easily hydrolyzed anhydride group, and four other conceivable metabolites in rats have been identified by GC-MS method in our previous study.AIM: To gain a clear idea of the cytochrome P450 enzyme isoforms associated with metabolism of norcantharidin.Methods Three dimensional structures of 8 CYP450 isoforms, including CYP1A2, CYP2A6,CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1 and CYP2C19 were established with homology modeling techniques, and then the CYP450s were investigated both through molecular docking with norcantharidin diacid and by experimental method using human recombinant CYP450s incubation system.Results The best enzyme-substrate structure obtained was CYP2El-norcantharidin diacid by comparing the docking parameters and analyzing the active sites of CYP2E1.This result was also supported by the in vitro metabolism study mediated by recombined human CYP2E1 enzyme using GC-MS for the determination of norcantharidin.Conclusion CYP4502E1 is the major isoform responsible for the oxidation metabolism of norcantharidin.The molecular docking method is useful for the prediction and mechanism research of drug metabolism.