Cytochrome P450 2B6 (CYP2B6), an important drug-metabolizing enzyme, is involved in the metabolism of prescribed drugs in humans. Despite its importance, cDNA for a CYP2B6 ortholog has not been identified and characterized in cynomolgus macaques, which are frequently used in preclinical studies. In this study, cDNA highly homologous to human CYP2B6 was cloned from the cynomolgus macaque liver. This cDNA contained an open reading frame of 491 amino acids and functional domains characteristic for CYP protein, such as substrate recognition sites and a heme-binding region. Cynomolgus CYP2B6 was expressed predominantly in the liver with some extra-hepatic expression among 10 tissues. Moreover, cynomolgus CYP2B6 revealed activities toward testosterone 16beta-hydroxylation and bupropion hydroxylation. These results suggest that cynomolgus CYP2B6 has a functional role in the liver.