Insulin brain resistant state is associated with cognitive deficits and Alzheimer's disease by mechanisms that may involve mitochondrial damage and oxidative stress. Conversely, physical exercise improves cognitive function and brain insulin signaling. The intracerebroventricular (i.c.v.) administration of streptozotocin (STZ) in rodents is an established model of insulin-resistant brain state. This study evaluates the effects of physical exercise on memory performance of i.c.v., STZ-treated mice(1 and 3 mg/kg) and whether insulin (50 and 100 ng/ml) modulates mitochondrial H₂O₂ generation in synaptosomes. S100B levels and SOD and CAT activities were assessed as markers of brain damage caused by STZ. Sedentary and exercise vehicle-treated mice demonstrated similar performance in object recognition memory task. In the water maze test, exercise vehicle-treated mice showed improvement performance in the acquisition and retrieval phases. The administration of STZ (1 mg/kg) before thirty days of voluntary physical exercise protocol impaired recognition and spatial memory only in exercised mice, whereas STZ (3 mg/kg) impaired the performance of sedentary and exercise groups. Moreover, STZ (3 mg/kg) increased hippocampal S100B levels in both groups and SOD/CAT ratio in the sedentary animals. Insulin decreased synaptosomal H₂O₂ production in exercised compared to sedentary mice; however, both STZ doses abolished this effect. Normal brain insulin signaling is mechanistically involved in the improvement of cognitive function induced by exercise through the regulation of mitochondrial H₂O₂ production. However, a prior blockade of brain insulin signaling with STZ abolished the benefits of exercise on memory performance and mitochondrial H₂O₂ regulation.