We investigated the metabolism of n-6 and n-3 fatty acids and their effect on lipid metabolism. Rat hepatocytes were a good model for the study of Δ5-desaturation, while HepG2 cells did not have this activity. We found that sesamin, a lignan of sesame seed, inhibited the activity of Δ5-desaturation in n-6 fatty acids but not in n-3 fatty acids. This result was confirmed in vivo using rats fed diets rich in n-6 and n-3 fatty acids. Sesamin also inhibited the increase of n-3 fatty acids in the liver caused by a n-3 fatty acid-rich diet. Sesamin had a unique function in changing the balance of intermediate metabolites of n-6 and n-3 fatty acids. We also examined the effect of fatty acids on lipid metabolism in HepG2 cells. Linolenic acid (LLA) decreased the synthesis and secretion of TG from HepG2 cells while linoleic acid (LA) and γ-linolenic acid (GLA) reduced only the secretion of TG. LA increased the binding and uptake of LDL. LLA had a strong effect in promoting the catabolism of cholesterol. These results suggest that a change in n-6/n-3 balance caused by a dietary component such as sesamin can affect lipid metabolism.