Ezetimibe inhibits intestinal cholesterol absorption, thereby reducing serum cholesterol. Recent studies suggest that ezetimibe affects liver steatosis and insulin resistance. We investigated the impact of ezetimibe on insulin sensitivity and glucose metabolism in C57BL/6 mice. We analyzed 4 mouse groups fed the following diets: normal chow (4% fat) for 12 weeks, normal chow for 10 weeks followed by normal chow plus ezetimibe for 2 weeks, high-fat chow (32% fat) for 12 weeks, and high-fat chow for 10 weeks followed by high-fat chow plus ezetimibe for 2 weeks. In the normal chow + ezetimibe group, ezetimibe had no impact on body weight, fat mass, lipid metabolism, liver steatosis, glucose tolerance, or insulin sensitivity. In the high-fat chow + ezetimibe group, ezetimibe had no impact on body weight or fat mass but significantly decreased serum low-density lipoprotein cholesterol, triglyceride, and glutamate pyruvate transaminase levels; liver weight; hepatic triglyceride content; and hepatic cholesterol content and increased the hepatic total bile acid content. In association with increases in IRS-2 and Akt phosphorylation, ezetimibe ameliorated hepatic insulin resistance in the high-fat chow + ezetimibe group, but had no effect on insulin sensitivity in primary cultured hepatocytes. A DNA microarray and Taqman polymerase chain reaction revealed that ezetimibe up-regulated hepatic SREBP2 and SHP expression and down-regulated hepatic SREBP-1c expression. SHP silencing mainly in the liver worsened insulin resistance, and ezetimibe protected against insulin resistance induced by down-regulation of SHP. Ezetimibe down-regulated SREBP-1c in the liver and reversed hepatic insulin resistance in mice fed a high-fat diet.