Aim To investigate the effects of ezetimibe on intracellular cholesterol level, and the mechanisms underlying the protective roles of ezetimibe against Chol: MβCD-induced cholesterol accumulation in vascular smooth muscle cells.Methods Vascular smooth muscle cells from rat were incubated with different concentrations of ezetimibe (3, 10, 30 μmol/L) or treated with 30 μmol/L ezetimibe for different time (6, 12, 24, 48 h).The intracellular cholesterol level was detected by high performance liquid chromatography,ezetimibe-regulated expression of LXRα, nSREBP-1 c, caveolin-1, and ABCA1 were analyzed by Western blot, RT-PCR, and indirect immunofluorescence.Results The intracellular free cholesterol, total cholesterol, and ratio of cellular cholesteryl ester to total cholesterol (CE/TC) were decreased.At the same time, the expression of caveolin-1, LXRα, and ABCA1 were enhanced in a dose-and time-dependent manner, but nSREBP-1c decreased.Conclusion The present study indicates that ezetimibe protects VSMCs from cholesterol accumulation by regulating the expression of lipid metabolism-related gene.Discussion We demonstrated that ezetimibe protects VSMCs from cholesterol accumulation, which might be related to the regulation of lipid metabolism-related gene expression and promotion of cholesterol efflux from VSMC foam cells.The regulation of two transcription factors, LXRα and SREBPs, is believed to be a key mechanism of how ezetimibe might mediate its functions, which resulting in an altered expression of corresponding target genes, such as ABCA1 and caveolin-1.This finding suggests that ezetimibe may inhibit the development of atherosclerosis or enhance its regression.