Abstract: The present study utilized LC-MS and HPLC approaches to characterize the metabolites of neferine in rat liver after an oral administration of 20 mg·kg^-1, and investigated the involvement of CYP450 isoforms in the metabolism of neferine by their selective inhibitors in vitro, separately. In positive ionization mode, besides neferine, four metabolites (M₁-M₄) were detected. M₂ (the major metabolite) and M₄ were identified as liensinine and isoliensinine by comparison with reference substances. Moreover, according to the analysis of metabolic rule of parent drug (neferine), M₁ and M₃ may be desmethyl-liensinine and desmethyl-isoliensinine, respectively. Furthermore, the metabolism of neferine in rat liver microsomes showed that the percentage inhibition of the major metabolism (liensinine) formation was 80.5% by quinidine (10 μmol·L^-1, selective CYP2D1 inhibitor) and 25.7% by ketoconazole (1 μmol·L^-1, selective CYP3A1 inhibitor). Neferine was mainly metabolized by CYP2D1 or CYP3A1 to liensinine, isoliensinine, desmethyl-liensinine and desmethyl-isoliensinine.