In this study, we identified the metabolites and the CYP forms that are specifically involved in emetine O-demethylation in human liver microsomes, and cleared the inhibitory potential of cephaeline and emetine on the activity of the major drug-metabolizing CYP enzymes. Incubation of emetine with human liver microsomes yielded three metabolites identified by using HPLC by comparison of the retention time with the authentic sample of cephaeline, 9-O-demethylemetine and 10-O-demethylemetine. CYP3A4 and CYP2D6 were able to metabolize emetine to cephaeline and 9-O-demethylemetine, and CYP3A4 also participated in metabolizing emetine to 10-O-demethylemetine. Cephaeline and emetine inhibited probe substrates metabolism. IC₅₀ for cephaeline against CYP2D6 and CYP3A4 were 121 and 1000 μM, respectively. For the emetine, CYP2D6 and CYP3A4 were 80 and 480 μM, respectively. Inhibition constants (K_i) for both compounds on the CYP2D6 and CYP3A4 activities were determined by graphic analysis of Dixon plots at various concentrations. The obtained K_i values of cephaeline for CYP2D6 and CYP3A4 were 54 and 355 μM, respectively, and the values of emetine were 43 and 232 μM, respectively. We concluded that these in vitro inhibitions of cephaeline and emetine would hardly increase plasma concentrations of co-administered drugs in clinical therapy.