Magnolin is a major bioactive component found in Shin-i, the dried flower buds of Magnolia fargesii; it has anti-inflammatory and anti-histaminic activities. Incubation of magnolin in human liver microsomes with an nicotinamide adenine dinucleotide phosphate-generating system resulted in the formation of five metabolites, namely, O-desmethyl magnolin (M1 and M2), didesmethylmagnolin (M3), and hydroxymagnolin (M4 and M5). In this study, we characterized the human liver cytochrome P450 (CYP) enzymes responsible for the biotransformation of three major metabolites--M1, M2, and M4--of magnolin. CYP2C8, CYP2C9, CYP2C19, and CYP3A4 were identified as the major enzymes responsible for the formation of the two O-desmethyl magnolins (M1 and M2), on the basis of a combination of correlation analysis and experiments, including immunoinhibition of magnolin in human liver microsomes and metabolism of magnolin by human cDNA-expressed CYP enzymes. CYP2C8 played a predominant role in the formation of hydroxymagnolin (M4). These results suggest that the pharmacokinetics of magnolin may not be affected by CYP2C8, CYP2C9, CYP2C19, and CYP3A4 responsible for the metabolism of magnolin or by the co-administration of appropriate CYP2C8, CYP2C9, CYP2C19, and CYP3A4 inhibitors or inducers due to the involvement of multiple CYP enzymes in the metabolism of magnolin.