Fish oil rich in n-3 polyunsaturated fatty acids has been shown to reduce the risk of cardiovascular diseases partly by reduction of blood triglyceride concentration. This favorable effect mainly results from the combined effects of inhibition of lipogenesis by decrease of SREBP-1 and stimulation of fatty acid oxidation by activation of peroxisome proliferator-activated receptor-α (PPARα) in liver. However, because fish oil is easily peroxidized to form hydroperoxides and increases oxidative stress, some defense mechanism(s) against oxidative stress might occur. To understand these complex effects of fish oil diet, the gene expression profile of mice liver was analyzed using high-density oligonucleotide arrays. High-fat diet (60% of total energy intake) as either safflower oil or fish oil (tuna) was given to mice. After 6 mo of feeding, expression levels of a total of 6,521 genes were analyzed. In fish oil diet compared with safflower oil diet, immune reaction-related genes, antioxidant genes (several glutathione transferases, uncoupling protein 2, and Mn-superoxide dismutase), and lipid catabolism-related genes upregulated, whereas cholesterol and fatty acid synthesis-related genes and 17-alpha hydroxylase/C17–20 lyase and sulfotransferases related to production of endogenous PPARα ligands and reactive oxygen species (ROS) downregulated markedly. Because upregulation of these antioxidant genes and downregulation of sulfotransferases were also observed in mice administered fenofibrate, altered gene expression related to antioxidant system observed in fish oil feeding was mediated directly and indirectly by PPARα activation. However, downregulation of 17-alpha hydroxylase/C17–20 lyase was not due to PPARα activation. These data indicate that fish oil feeding downregulated the endogenous PPARα-activation system and increased antioxidant gene expressions to protect against ROS excess.