Aim To investigate the effects of long-term ezetimibe on hyperlipidemia and genes involved in lipid metabolism in the liver of the LDLR＋/－ hamster. Methods Male LDLR＋/－ hamsters were randomly divided into two groups:the vehicle group and the ezetimibe group (25 mg/(kg·d)). Animals were free to access water and high-fat diet. After 20-week administration, hamsters were sampled after overnight fasting. The plasma levels of total cholesterol (TC), triglyceride (TG), total bile acid (TBA), non-esterified fatty acid (NEFA) and aspartate aminotransferase (AST) were detected by assay kits. Mixed plasma in each group was further separated via an KTA fast protein liquid chromatography (FPLC) system and the TC and TG levels in each fraction were also detected. The expression of multiple genes in the liver and proteins in the plasma was determined by qRT-PCR and Western blot, respectively. Results Long-term ezetimibe intervention significantly decreased plasma TC, TG, TBA, NEFA and AST levels, and apolipoprotein B protein expression in the LDLR＋/－hamsters, and had no effect on the protein levels of lipoprotein lipase (LPL) and proprotein convertase subtilisin/kexin-type 9 (PCSK9) and the activity of LPL. Further KTA-FPLC analysis demonstrated that ezetimibe reduced the TC and TG levels in all the lipoprotein fractions. In the liver, ezetimibe significantly reduced TC, but not TG concentration. Furthermore, ezetimibe significantly increased the mRNA expression of sterol regulatory element binding protein 2 and PCSK9 and dramatically decreased the expression of liver X receptor α, cholesterol 7α-hydroxylase A1, ATP-binding cassette transporter G5 (ABCG5) and ABCG8 in the liver. Conclusion Long-term ezetimibe intervention reduces hyperlipidemia and displays complex modulatory effects on the genes involved in lipid homeostasis in LDLR＋/－ hamsters.