Abstract: This study evaluated the influence of melatonin on cholesterol absorption in rats fed on high cholesterol diet (HCD). HCD induced a remarkable increase in hepatic and plasma total cholesterol, plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol, a decrease in high density lipoprotein (HDL) cholesterol and an elevation in triacylglyceride (TG) levels in plasma and in the liver. Melatonin suspension (10 mg/kg), specially prepared for this purpose, cholestyramine (230 mg/kg) and ezetimibe (145 μg/kg) were administered orally to the rats fed HCD for 30 days. Melatonin significantly reduced cholesterol absorption in rats fed on HCD and caused significant decreases in total cholesterol, TG, VLDL- and LDL-cholesterol in the plasma and contents of cholesterol and TG in the liver. The level of HDL cholesterol was significantly increased after melatonin. These results suggested that inhibition of cholesterol absorption caused by melatonin could be a mechanism contributing to the positive changes in plasma cholesterol, lipoprotein profile and the lipid contents in the liver.