Polyphenol-rich grape seeds have a beneficial effect on human health. The present study was performed to investigate the effects of grape seeds on antioxidant activities in rats. Male Sprague-Dawley rats were randomly divided into a control diet group (C), a high-fat diet group (HF), a 5% grape seed-supplemented control diet group (G), and a 5% grape seed-supplemented high-fat diet group (HG). Dietary supplementation with grape seeds reduced serum concentrations of lipid peroxides compared with those in the C and HF groups. The hepatic level of lipid peroxides decreased significantly in the grape seed groups compared with that in the C and HF groups. Superoxide dismutase activity in the G group increased significantly compared with that in the C group. Catalase activity tended to be higher by feeding grape seeds. The grape seed diet increased glutathione peroxidase activity in the C group. Glutathione-S-transferase activity increased significantly in the G group compared with that in the C group. Hepatic content of total glutathione increased significantly in the HG group but decreased significantly in the HF group. The ratio of reduced glutathione and oxidized glutathione increased by feeding the grape seed diet. Total vitamin A concentration was significantly higher in HG group than in other groups. Liver tocopherol content of the G and HG groups was significantly higher than that of the control groups. These results suggest that dietary supplementation with grape seeds is beneficial for suppressing lipid peroxidation in high fat-fed rats.