Obesity is an endemic problem in the United States and elsewhere, and data indicate that in addition to overconsumption, exposure to specific chemicals enhances obesity. CYP2B metabolizes multiple endo- and xenobiotics, and recent data suggests that repression of Cyp2b activity increases dyslipidemia and age-onset obesity, especially in males. To investigate the role played by Cyp2b in lipid homeostasis and obesity, we treated wildtype and Cyp2b-null mice with a normal (ND) or 60% high-fat diet (HFD) for 10 weeks and determined metabolic and molecular changes. Male HFD-fed Cyp2b-null mice weigh 15% more than HFD-fed wildtype mice, primarily due to an increase in white adipose tissue (WAT); however, Cyp2b-null female mice did not demonstrate greater body mass or WAT. Serum parameters indicate increased ketosis, leptin and cholesterol in HFD-fed Cyp2b-null male mice compared to HFD-fed wildtype mice. Liver triglycerides and liver:serum triglyceride ratios were higher than their similarly treated wildtype counterparts in Cyp2b-null male mice, indicating a role for Cyp2b in fatty acid metabolism regardless of diet. Furthermore, RNAseq demonstrates that hepatic gene expression in ND-fed Cyp2b-null male mice is similar to HFD-fed WT male mice, suggestive of fatty liver disease progression and a role for Cyp2b in lipid homeostasis. Females did not show as demonstrative changes in liver health, and significantly fewer changes in gene expression, as well as gene expression associated with liver disease. Overall our data indicates that the repression or inhibition of CYP2B may exacerbate metabolic disorders and cause obesity by perturbing fatty acid metabolism, especially in males.