Cinobufagin (CB), a major bioactive component of the traditional Chinese medicine Chansu, has been reported to have potent antitumor activity. In this study, in vitro metabolism of CB among species was compared with respect to metabolic profiles, enzymes involved, and catalytic efficiency by using liver microsomes from human (HLM), mouse (MLM), rat (RLM), dog (DLM), minipig (PLM), and monkey (CyLM). Significant species differences in CB metabolism were revealed. In particular, species-specific deacetylation and epimerization combined with hydroxylation existed in RLM, whereas hydroxylation was a major pathway in HLM, MLM, DLM, PLM, and CyLM. Two monohydroxylated metabolites of CB in human and animal species were identified as 1α-hydroxylcinobufagin and 5β-hydroxylcinobufagin by using liquid chromatography-mass spectrometry and two-dimensional NMR techniques. CYP3A4 was identified as the main isoform involved in CB hydroxylation in HLM on the basis of the chemical inhibition studies and screen assays with recombinant human cytochrome P450s. Furthermore, ketoconazole, a specific inhibitor of CYP3A, strongly inhibited CB hydroxylation in MLM, DLM, PLM, and CyLM, indicating that CYP3A was responsible for CB hydroxylation in these animal species. The apparent substrate affinity and catalytic efficiency for 1α- and 5β-hydroxylation of CB in liver microsomes from various species were also determined. PLM appears to have K(m) and total intrinsic clearance value (V(max)/K(m)) similar to those for HLM, and the total microsomal intrinsic clearance values for CB obeyed the following order: mouse > dog > monkey > human > minipig. These findings provide vital information to better understand the metabolic behaviors of CB among various species.