This study investigated the neuroprotective effects of dietary supplementation of fish oil on both brain infarction and the activities of antioxidant enzymes. Male Sprague-Dawley rats (4-weeks old) were divided into two groups and received either a regular diet (RD) or a fish-oil-supplemented diet (FOD) for 6 weeks prior to middle cerebral artery (MCA) occlusion. The infarction volume of the brain was calculated using image analysis after staining. Antioxidant enzymes were measured before ischemia (BI), after 2 h of ischemia (AI) and after 24 h (24hR), 48 h (48hR) and after 7 days (7dR) of reperfusion. The infarction volume of the brain was significantly smaller in the FOD group than in the RD group after 24 h of reperfusion (p<0.05). Before ischemia, the levels of lipid peroxide and the glutathione peroxidase (GPx) activity were higher in the FOD group than in the RD group. During reperfusion, the catalase (CAT) activity in the FOD group remained at the preischemia level until after 48 h of reperfusion, while those in the RD group did not. The Mn-superoxide dismutase (SOD) activity and GPx activity were higher in the FOD group than in the RD group only after 2 h of ischemia. In the fatty acid analysis, the ratio of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were higher in the FOD group than in the RD group (p<0.05). Our results demonstrate that supplementing the diet with fish oil could decrease the cerebral infarction volume following ischemia and reperfusion (I/R) partly by working directly as an antioxidant and partly by modulating antioxidant enzyme activities.