There is currently no established treatment for non-alcoholic fatty liver disease (NAFLD), including its most extreme form, non-alcoholic steatohepatitis (NASH). Ezetimibe, an inhibitor of Niemann-Pick C1 Like 1-dependent cholesterol absorption, improves diet-induced hyperlipidemia and attenuates liver steatosis and insulin resistance. The aim of the present study was to determine whether ezetimibe treatment is able to inhibit the development of NAFLD, and to elucidate the underlying mechanism, using C57BL/6J (B6) mice maintained on a high-fat diet. Male B6 mice (20 weeks of age) were divided into the following two groups (n=7 in each group): Mice fed a high-fat diet for four weeks and mice fed a high-fat diet with 0.0064% (wt/wt) ezetimibe (5 mg/kg/day) for four weeks. Administration of ezetimibe significantly reduced liver steatosis and fibrosis. Ezetimibe reduced serum cholesterol, hepatic fat accumulation and insulin resistance in the liver of mice fed the high-fat diet. Furthermore, ezetimibe significantly reduced hepatic mRNA expression of Acc1 and Scd1, which are involved in hepatic fatty acid synthesis. Ezetimibe significantly reduced hepatic Cd36 gene expression, upregulation of which is significantly associated with insulin resistance, hyperinsulinemia and increased steatosis. The protein expression of SKP2, a viable therapeutic target in human cancer, was also reduced by ezetimibe. These findings suggest that ezetimibe may be an effective therapy for high fat-induced NAFLD, including NASH.