Human cytochrome P4502B6 (CYP2B6) is predominantly expressed in the liver and it plays a major role in the metabolism of several therapeutically important drugs and environmental toxicants. The objective was twofold: (1) to determine the role of genetic, physiological, and environmental factors in predicting hepatic CYP2B6 protein expression; and (2) to investigate the role of CYP2B6 in nicotine C-oxidation. Human livers (n = 40) were assessed for CYP2B6 protein and genotype. Linear regression analyses indicated that CYP2B6 genotype (10%), gender (14%), and exposure to inducers (21%), but not age, were predictors of CYP2B6 protein amounts. Livers with at least one CYP2B6*5 or *6 allele were associated with lower CYP2B6. Female livers and livers exposed to inducers (phenobarbital and/or dexamethasone) were associated with higher CYP2B6. A weak correlation between CYP2B6 and nicotine C-oxidation activity was observed, which was abrogated when controlling for CYP2A6 protein levels. CYP2B6*6 was not associated with different nicotine kinetics. In summary, CYP2B6 protein expression was associated with genotype, gender, and exposure to inducers, but not with nicotine C-oxidation activity.