We have reported that cytochrome P450 (CYP) 1B1 contributes to hypertension in various experimental models and promotes neointimal growth in injured carotid arteries. This study was conducted to determine the role of CYP1B1 in the development of atherosclerosis and its pathogenesis, including reactive oxygen species (ROS) production, inflammation, and hypertension caused by atherogenic diet (AD; TD02028). Eight weeks old male ApoE knockout (ApoE-/-/Cyp1b1+/+) and ApoE /CYP1B1 knockout (ApoE-/-/Cyp1b1-/-) mice were fed a normal diet (ND) or AD for 12 weeks. A separate group of ApoE-/-/Cyp1b1+/+ mice on AD, were injected twice weekly with the selective inhibitor of CYP1B1, 2,3',4,5'-tetramethoxystilbene (TMS) (300 μg/kg), or its vehicle DMSO (i.p.). Systolic blood pressure (SBP) was measured weekly by tail cuff. After 12 weeks, animals were sacrificed; blood was collected for lipid analysis and aorta for measurement of lesions by Oil red O, collagen deposition by Masson’s trichrome and ROS production by dihydroethidium staining, and infiltration of macrophages (F4/80) and T-lymphocytes (CD3) by immunohistochemistry. Plasma lipid levels, area of lipids and collagen deposition, ROS production (Table), infiltration of macrophages, and T lymphocytes in the aorta and SBP were increased in ApoE-/-/Cyp1b1+/+ mice on AD vs. ND; these were minimized in mice on AD given TMS but not its vehicle, and in ApoE-/-/Cyp1b1-/- mice on AD. These data suggest that the development of hypercholesterolemia by AD and associated pathogenesis including increase in SBP in ApoE-/-/Cyp1b1+/+ mice depend on CYP1B1 and that it could serve as a novel target for treatment of atherosclerosis.