Corydaline is the bioactive alkaloid isolated from Corydalis Tubers with various antiacetylcholinesterase, antiallergic, and antinociceptive activities. The purposes of this study were to identify the in vitro metabolism of corydaline in human liver microsomes and to characterize the cytochrome P450 (CYP) enzymes responsible for the metabolism of corydaline. Incubation of corydaline with human liver microsomes in the presence of an NADPH generating system resulted in the formation of eight metabolites including O-desmethylcorydaline (M1, M2, M3, and M4), hydroxydesmethylcorydaline (M5), didesmethylcorydaline (M6, M7), and hydroxycorydaline (M8). The formation of desmethylcorydaline, M1 from corydaline was major metabolic pathway, and the formation of desmethylcorydaline, M2, M3, and M4 and hydroxycorydaline (M8) were minor metabolic pathways. The human CYP enzymes responsible for corydaline metabolism were characterized by evaluating on the basis of a combination of correlation analysis and experiments including immunoinhibition of corydaline in human liver microsomes and metabolism of corydaline by human cDNA-expressed CYP enzymes. CYP2C9 and CYP3A4 were major enzymes involved in the formation of a major metabolite, O-desmethylcorydaline, M1 from corydaline. CYP3A4 was the major enzyme for the formation of O-desmethylcorydaline, M2 and M3, and hydroxycorydaline, M8 from corydaline with a little involvement of CYP2D6, CYP1A2, or CYP2C8, respectively. These results suggest that the pharmacokinetics of corydaline may be affected by CYP2C9 and CYP3A4 responsible for the metabolism of corydaline or by the co-administration of appropriate CYP2C9 and CYP3A4 inhibitors or inducers.