The Cyp2b subfamily contains five members (Cyp2b9, Cyp2b10, Cyp2b13, Cyp2b19, and Cyp2b23) of which three (Cyp2b9, Cyp2b10, Cyp2b13) are hepatic enzymes involved in xenobiotic detoxification. We made a Cyp2b-knockdown mouse using lentiviral-promoted shRNA homologous to all five Cyp2b subfamily members to characterize Cyp2b’s role in xenobiotic detoxification. Western blots from 8-week old mice demonstrate that hepatic Cyp2b’s are repressed 5-10X by the shRNA. Unexpectedly, Cyp2b-KD mice showed significant changes in some organ weights, especially an increase in abdominal, inguinal, and renal adipose. Both male and female Cyp2b-KD mice showed significant increase in fat to body weight ratio (112.1% and 73.5% for males and females, respectively). Interestingly, associated with changes in fat to body ratios was changes in non-fasting triglycerides levels. Non-fasting serum triglyceride was increased 18-39% in 8-10 weeks old female and male mice, respectively with a statistically significant increase in males. Therefore, wild-type and Cyp2b-knockdown mice were housed for 35 weeks and necropsies performed to test whether older Cyp2b-knockdown mice show perturbed lipid utilization. Thirty-five week old Cyp2b-KD mice exhibited a 17 to 22% increase in their body weight caused by a significant increase in fat deposition compared to wild-type (FVB) mice. This was accompanied by increased triglyceride and low density lipoprotein levels. Subsequent studies were performed with 100ml of corn oil or the CAR activator and CYP-inducer TCPOBOP at 3mg/kg/ in corn oil as a carrier. Surprisingly, 8-12 weeks Cyp2b-knockdown mice showed some difficulty in dealing with the corn oil. Significant increases in total serum cholesterol (25-39%) was observed in the corn oil control Cyp2b-knockdown male and female mice, respectively, compared to the WT mice, probably because the Cyp2b-KD mice were unable to respond to the unsaturated fatty acids in the corn oil. The increases in non-fasting serum total cholesterol and triglyceride was reversed by TCPOBOP treatment in Cyp2b-knockdown male mice but not in Cyp2bknockdown female mice as TCPOBOP treatment caused a significant increase in triglyceride in Cyp2b-KD females compared to WT mice. Cyp2b-KD mice appear to have perturbations in clearing corn oil from the liver as corn oil treatment increased Oil Red O staining indicating accumulation of lipids in the livers of Cyp2b-KD mice compared to WT mice. FoxA2 and Cpt1a, involved in triglyceride homeostasis, were increased 2-6 fold, albeit not significantly, in Cyp2p-KD mice treated with corn oil. In conclusion, changes in Cyp2b expression led to perturbation in lipid metabolism in Cyp2b-KD mice. This suggests that Cyp2b is more than a detoxification enzyme, but is also involved in the metabolism of unsaturated fatty acids, as Cyp2b-KD mice have increased fat deposition and show increased serum and liver lipid levels.