Eperisone is a centrally acting muscle relaxant widely used for the therapeutic treatment of spastic patients to relieve muscle stiffness and back pain. The objective of this study was to characterize the metabolic pathway involved in the biotransformation of eperisone mediated by human cytochrome P450 (CYP) enzymes. Eperisone was metabolized to seven metabolites via oxidation and carbonyl reduction in human liver microsome. Among them, M3 and M4 were found to be primary major metabolites which were generated by CYPs. The kinetics study with (-)-R- and (+)-S-eperisones revealed that CYPs-mediated hydroxylation did not have significant stereoselectivity for metabolic clearance of eperisone. Incubation with recombinant CYP isozyme, chemical inhibition assay, and immuno-inhibition assay showed that multiple CYPs were involved in M4 formation, but mainly CYP2J2 in M3 formation. In addition, intestinal microsomes metabolized eperisone to M3 and M4 via CYP2J2- and CYP3A4-mediated reactions, which are supposed to contribute to presystemic metabolism of eperisone.