Sesamin is a specific inhibitor of delta 5 desaturation, the conversion from dihomo-gamma-linolenic acid (20:3, n-6) to arachidonic acid (AA, 20:4, n-6). Previously, we reported that sesamin inhibited delta 5 desaturation of n-6 fatty acids in rat hepatocytes but not that of n-3 fatty acids, from 20:4 (n-3) to eicosapentaenoic acid (EPA, 20:5, n-3). In this study, we investigated the interaction of sesamin and EPA on delta 5 desaturation of both series and the n-6/n-3 fatty acids ratio by measuring actural fatty acid contents in vivo. Rats were fed three types of dietary oils; 1) linoleic acid (LA, 18:2, n-6): linolenic acid (LLA, 18:3, n-3) = 3:1, n-6/n-3 ratio of 3:1 (LA group), 2) LA:LLA = 1:3, n-6/n-3 ratio of 1:3 (LLA group), 3) LA:LLA:EPA = 1:0.5:3, n-6/n-3 ratio of 1:3.5 (EPA group) with or without sesamin (0.5% w/w) for 4 weeks. In all groups, sesamin administration increased the content of dihomo-gamma-linolenic acid (20:3, n-6) in the liver and decreased the delta 5 desaturation index of n-6 fatty acid, the ratio of 20:4/20:3 (n-6). On the contrary, the delta 5 desaturation index of n-3 fatty acid, the ratio of 20:5 + 22:5 + 22:6/20:4 (n-3), was increased by the administration of sesamin. These results suggest that sesamin inhibits the delta 5 desaturation of n-6 fatty acid, but not that of n-3 fatty acid in rat livers. Sesamin administration decreased incorporation of EPA (n-3) and simultaneously increased the AA (n-6) content in the liver. The n-6/n-3 ratio in the liver was increased by administering sesamin under n-3 rich conditions, i.e., the LLA and EPA groups.