The aim of the present study was to identify molecular mechanisms involved in liver fat and cholesterol accumulation in ovariectomised (Ovx) rats fed with high-cholesterol diets. VLDL assembly and bile acid metabolism were specifically targeted. After being either Ovx or sham-operated, the rats were fed a standard diet or a high-fat diet containing 0, 0·25 or 0·5 % cholesterol for 6 weeks. Although Ovx rats exposed to dietary cholesterol intake accumulated the greatest amount of hepatic fat and cholesterol, plasma cholesterol levels were lower (P< 0·05) in these animals than in the corresponding control rats. Accompanying this observation, ovariectomy and dietary cholesterol intake resulted in a down-regulation (P< 0·05) of the expression of genes associated with VLDL assembly, including microsomal TAG transfer protein, diacylglycerol acyltransferase 2, acyl-CoA:cholesterol acyltransferase 2 and apoB-100 as well as genes associated with bile acid metabolism including farnesoid X receptor and bile salt export pump (P< 0·01). These results indicate that high-fat/high-cholesterol diets and ovariectomy concomitantly disrupt hepatic lipid output through defects in VLDL assembly and, most probably, secretion. The results also point to a defect in hepatic bile acid secretion. The present study offers novel insights into intrahepatic lipid metabolism, which may be relevant to metabolic complications found in postmenopausal women.