Linoleic acid (LA) has a two-sided effect with regard to serum cholesterol-lowering and pro-inflammation, although whether this fatty acid reduces serum cholesterol and the development of atherosclerosis under high-cholesterol conditions has yet to be ascertained. In this study, we examine the effects of dietary LA on reducing serum cholesterol and atherosclerosis development under high-cholesterol conditions. Male and female apoE-deficient (ApoE-/-) mice were fed AIN-76-based diets containing 10% SFA and 0·04 % cholesterol, 10% LA and 0·04% low cholesterol (LALC), or 10% LA and 0·1% high cholesterol (LAHC) for 9 weeks. The results revealed significant reduction in serum cholesterol levels and aortic lesions with increasing levels of pro-inflammatory biomarkers (urinary isoprostane and aortic MCP-1 mRNA) in male and female LALC groups compared with those in the SFA groups (P < 0·05). Furthermore, whereas there were significant increases in the serum cholesterol levels and aortic lesions (P < 0·05), there was no difference in aortic MCP-1 mRNA levels in male and female LAHC groups compared with those in the LALC groups. A high-dietary intake of cholesterol eliminated the serum cholesterol-lowering activity of LA but had no significant effect on aortic inflammation in either male or female ApoE-/- mice. The inhibitory effect of LA on arteriosclerosis is cancelled by a high-cholesterol diet due to a direct increase in serum cholesterol levels. Accordingly, serum cholesterol levels might represent a more prominent pathogenic factor than aortic inflammation in promoting the development of atherosclerosis.