Several studies have shown that mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) opening prevents inchemia/reperfusion injuries in heart. However, how mitoK_ATP is opened and its effects in brain are still unclear. In this study we have tried to verify if mitochondrial reactive oxygen species (ROS) could activate mitoK_ATP and some of its effects on oxidative phosphorylation and membrane potential in isolated brain rat mitochondria. Using endogenous ROS produced by mitochondrial reverse electron transport we found that mitoK_ATP could be activated by ROS, in a manner prevented by rotenone. In this context, exogenous H₂O₂ also activated swelling secundary to K⁺ entrance, in a manner prevented by catalase. Both processes were inhibited by ATP, demonstrating that these effects were due to mitoK_ATP. No significant changes in either membrane potential nor oxidative phosphorylation were observed in brain mitochondria upon mitoK_ATP activation. In cultured neurons, mitoK_ATP activation prevented cell death and oxidative stress promoted by N-methyl D-aspartate (NMDA). Together, our results suggest that brain mitoK_ATP acts as a ROS sensor and prevents oxidative stress under pathological conditions.