Partially inbred lines of laboratory opossums differ in plasma low-density lipoprotein cholesterol concentration and cholesterol absorption on a high-cholesterol diet. The aim of the present studies was to determine whether ezetimibe inhibits cholesterol absorption and eliminates the differences in plasma cholesterol and hepatic cholesterol metabolism between high and low responders on a high-cholesterol diet. Initially, we determined that the optimum dose of ezetimibe was 5 mg/(kg d) and treated 6 high- and 6 low-responding opossums with this dose (with equal numbers of controls) for 3 weeks while the opossums consumed a high-cholesterol and low-fat diet. Plasma and low-density lipoprotein cholesterol concentrations decreased significantly (P < .05) in treated but not in untreated high-responding opossums. Plasma cholesterol concentrations increased slightly (P < .05) in untreated low responders but not in treated low responders. The percentage of cholesterol absorption was significantly higher in untreated high responders than in other groups. Livers from high responders with or without treatment were significantly (P < .01) heavier than livers from low responders with or without treatment. Hepatic cholesterol concentrations in untreated high responders were significantly (P < .05) higher than those in low responders with or without treatment (P < .001). The gall bladder bile cholesterol concentrations in untreated high responders were significantly (P < .05) lower than those in other groups. A decrease in biliary cholesterol in low responders treated with ezetimibe was associated with a decrease in hepatic expression of ABCG5 and ABCG8. These studies suggest that ezetimibe decreases plasma cholesterol levels in high responders mainly by decreasing cholesterol absorption and increasing biliary cholesterol concentrations. Because ezetimibe's target is NPC1L1 and NPC1L1 is expressed in the intestine of opossums, its effect on cholesterol absorption may be mediated by inhibiting NPC1L1 function in the intestine.