One of the major risk factors for ischemic disease is hyperlipidemia, which is mainly regulated by endogenous cholesterol synthesis in the liver and dietary absorption in the small intestine. In this study, we evaluated the vascular protective effects of a potent cholesterol absorption inhibitor, ezetimibe. ApoE-deficient mice were fed a chow or high-fat diet with or without ezetimibe (5mg/(kgday)) for 3 months. Co-treatment with ezetimibe significantly reduced plasma cholesterol (by 76%; from 1592 to 381mg/dL) and LDL cholesterol (by 78%; from 1515 to 319mg/dL), and increased HDL cholesterol (by 187%; from 16 to 46mg/dL) in high-fat diet mice. Consistently, a marked inhibitory effect of ezetimibe on the development of lipid-rich plaque was observed, as assessed by oil red O staining. Of importance, treatment with ezetimibe significantly improved endothelial dysfunction as assessed by the vasodilator response to acetylcholine, accompanied by inhibition of interleukin-6 mRNA and an increase in endothelial nitric oxide synthase (eNOS) mRNA in the aorta. Ezetimibe also suppressed oxidative stress and the ubiquitination-proteasome system in the aorta. Although changes in body weight and several tissue weights were similar in the groups with and without ezetimibe administration, only liver weight was significantly decreased in the ezetimibe-treated group. Interestingly, ezetimibe markedly inhibited lipid accumulation in the liver. Furthermore, ezetimibe increased the mRNA expression of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase as a counteraction in the liver, but not in the aorta. Overall, ezetimibe significantly prevented atherosclerosis through not only lipid-lowering effects, but also other direct and/or indirect vascular protective actions in ApoE-deficient mice.