This work investigated the ability of melatonin to prevent oxidative damage in brain tissue induced by injection of beta-amyloid peptide 25–35 (Aβ25–35) in middle-aged rats. The Morris water maze was used to evaluate the cognitive function of the rats. Thiobarbituric acid-reactive substances and antioxidative enzymes (superoxide dismutase and glutathione peroxidase) activities were measured. It was found that injection of (Aβ25–35) (20 μg) into the rat hippocampus caused an increase in the latency (the time to find the platform), the total swimming distance to the platform, and the starting angles in (Aβ25–35)-treated rats. Furthermore, a significant rise in lipid peroxidation and decrease in antioxidative enzyme activities in brain tissue were found. Melatonin (0.1, 1, and 10 mg/kg, i.g. × 10 days) improved the spatial resolution of amnesic rats in the Morris water maze test. Meanwhile, melatonin antagonized the lipid peroxidation in both the mitochondria (P < 0.01) at the doses of 0.1, 1.0, and 10 mg/kg and in the cytoplasm at the doses of 0.1 and 1.0 mg/kg. Also in the amnesic rats, melatonin (0.1, 1.0, and 10 mg/kg, i.g. × 10 days) stimulated the antioxidative enzyme activities. The results show that melatonin effectively reduced lipid peroxidation and enhanced the antioxidative enzyme activities in Aβ25–35-treated rats, which may contribute to the improvement of rats' learning and memory impaired by Aβ25–35.