The effects of sesamin, one of the most abundant lignans in sesame seed, on hepatic fatty acid oxidation were examined in rats that were fed experimental diets containing various amounts (0%, 0.1%, 0.2%, and 0.5%) of sesamin (a 1:1 mixture of sesamin and episesamin) for 15 days. Dietary sesamin dose-dependently increased both mitochondrial and peroxisomal palmitoyl-coenzyme A (CoA) oxidation rates. Mitochondrial activity almost doubled in rats on the 0.5% sesamin diet. Peroxisomal activity increased more than 10-fold in rats fed a 0.5% sesamin diet in relation to rats on the sesamin-free diet. Dietary sesamin greatly increased the hepatic activity of fatty acid oxidation enzymes, including carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase. Dietary sesamin also increased the activity of 2,4-dienoyl-CoA reductase and delta3,delta2-enoyl-CoA isomerase, enzymes involved in the auxiliary pathway for beta-oxidation of unsaturated fatty acids dose-dependently. Examination of hepatic mRNA levels using specific cDNA probes showed a sesamin-induced increase in the gene expression of mitochondrial and peroxisomal fatty acid oxidation enzymes. Among these various enzymes, peroxisomal acyl-CoA oxidase and bifunctional enzyme gene expression were affected most by dietary sesamin (15- and 50-fold increase by the 0.5% dietary level). Sesamin-induced alterations in the activity and gene expression of carnitine palmitoyltransferase I and acyl-CoA oxidase were in parallel with changes in the mitochondrial and peroxisomal palmitoyl-CoA oxidation rate, respectively. In contrast, dietary sesamin decreased the hepatic activity and mRNA abundance of fatty acid synthase and pyruvate kinase, the lipogenic enzymes. However, this lignan increased the activity and gene expression of malic enzyme, another lipogenic enzyme. An alteration in hepatic fatty acid metabolism may therefore account for the serum lipid-lowering effect of sesamin in the rat.