Common variation in the CYP2B6 gene, encoding the cytochrome P450 2B6 enzyme, is associated with substrate-specific altered clearance of multiple drugs. CYP2B6 is a minor contributor to hepatic nicotine metabolism, but the enzyme has been proposed as relevant to nicotine-related behaviors because of reported CYP2B6 mRNA expression in human brain tissue. Therefore, we hypothesized that CYP2B6 variants would be associated with altered nicotine oxidation, and that nicotine metabolism by CYP2B6 would be detected in human brain microsomes. We generated recombinant enzymes in insect cells corresponding to nine common CYP2B6 haplotypes and demonstrate genetically determined differences in nicotine oxidation to nicotine iminium ion and nornicotine for both (S) and (R)-nicotine. Notably, the CYP2B6.6 and CYP2B6.9 variants demonstrated lower intrinsic clearance relative to the reference enzyme, CYP2B6.1. In the presence of human brain microsomes, along with nicotine-N-oxidation, we also detect nicotine oxidation to nicotine iminium ion. However, unlike N-oxidation, this activity is NADPH independent, does not follow Michaelis-Menten kinetics, and is not inhibited by NADP or carbon monoxide. Furthermore, metabolism of common CYP2B6 probe substrates, methadone and ketamine, is not detected in the presence of brain microsomes. We conclude that CYP2B6 metabolizes nicotine stereoselectively and common CYP2B6 variants differ in nicotine metabolism activity, but did not find evidence of CYP2B6 activity in human brain.