The mitochondrial electron transport system (ETS) is a main source of cellular ROS, including hydrogen peroxide (H2O2). The production of H2O2 also involves the mitochondrial membrane potential (ΔΨm) and oxygen consumption. Impaired insulin signaling causes oxidative neuronal damage and places the brain at risk of neurodegeneration. We evaluated whether insulin signaling cross-talk with ETS components (complexes I and FoF1ATP synthase) and ΔΨm to regulate mitochondrial H2O2 production, in tissue preparations from rat brain. Insulin (50 to 100ng/ml) decreased H2O2 production in synaptosomal preparations in high Na(+) buffer (polarized state), stimulated by glucose and pyruvate, without affecting the oxygen consumption. In addition, insulin (10 to 100ng/ml) decreased H2O2 production induced by succinate in synaptosomes in high K(+) (depolarized state), whereas wortmannin and LY290042, inhibitors of the PI3K pathway, reversed this effect; heated insulin had no effect. Insulin decreased H2O2 production when complexes I and FoF1ATP synthase were inhibited by rotenone and olygomicin respectively suggesting a target effect on complex III. Also, insulin prevented the generation of maximum level of ∆Ѱm induced by succinate. The PI3K inhibitors and heated insulin maintained the maximum level of ∆Ѱm induced by succinate in synaptosomes in a depolarized state. Similarly, insulin decreased ROS production in neuronal cultures. In mitochondrial preparations, insulin neither modulated H2O2 production or oxygen consumption. In conclusion, the normal downstream insulin receptor signaling is necessary to regulate complex III of ETS avoiding the generation of maximal ∆Ѱm and increased mitochondrial H2O2 production.