Multiple factors in addition to overconsumption are believed to lead to obe-sity and non-alcoholic fatty liver disease (NAFLD) in the United States and elsewhere. Cyp2b-null mice that lack the primarily hepatic Cyp2b enzymes are diet-induced obese, primarily due to increased white adipose tissue, but also contain increased liver triglycerides. Therefore, studies were undertaken to determine whether human CYP2B6 is anti-obesogenic and a putative mechanism. Vivid human CYP2B6 recombinant enzyme was used to determine inhibitors of CYP2B6. Several polyunsaturated fatty acids (PUFAs) including arachidonic acid, linoleic acid, DHA, and α-linolenic acid (ALA) were inhibitors with EC50’s below 10μM. LC-MS/MS revealed that CYP2B6 metabolized all PUFAs tested; however, there was a greater than 20-fold preference for metabolism of to 9-HOTre and to a lesser extent 13-HOTre, monohydroxylated products of ALA. This data indicates that CYP2B6 metabolizes a specific PUFA (α-linolenic acid) to a specific metabolite (9-HOTre) with unknown function. Interestingly, cellular incorporation of Nile Red, a triglyceride sensor, is greater in ALA-treated CYP2B6-transfected HepG2 cells than untransfected HepG2 cells, indicating that metabolism of ALA by CYP2B6 increases hepatic lipid accumulation, the opposite of what was expected. Increased lipid localization in ALA-treated CYP2B6-HepG2 cells was confirmed and quantified using confocal microscopy. We recently developed a humanized CYP2B6 mouse on our Cyp2b-null background and provided a 60% high-fat diet to hCYP2B6-Tg and Cyp2b-null mice for 16 weeks. The presence of CYP2B6 reduced weight gain and metabolic disease as measured by glucose tolerance tests, how-ever hCYP2B6-Tg male mice showed increased liver triglycerides, consistent with the data described in the CYP2B6-HepG2 cells. Currently, we are identifying changes in serum oxylipins and hepatic ALA metabolism between hCYP2B6-Tg and Cyp2b-null mice. In addition, several pharmaceuticals and environmental chemicals were screened using the Vivid CYP2B6 recombinant enzyme. Triclosan, diazinon, endosulfan, atrazine, ticlopidine, parathion, chlorpyrifos, jet fuel, nonylphenol, and others were found to be CYP2B6 inhibitors. Overall, this study defines a mechanism by which chemical inhibition of CYP2B6 can increase diet-induced obesity and metabolic disease through reduced production of ALA metabolites and disruption of lipid distribution between the liver and other tissues.