This study investigated the cytoprotective effect of butin, a flavonoid, on hydrogen peroxide (H2O2)-induced mitochondrial dysfunction. Electron spin resonance (ESR) spectrometry revealed butin’s significant scavenging effects on superoxide radicals and hydroxyl radicals. When H2O2 was used to induce an increase in mitochondrial reactive oxygen species (ROS) in Chinese hamster lung fibroblast (V79-4) cells, butin treatment decreased high level of ROS. Butin also attenuated intracellular Ca2+ levels that have been induced by H2O2. Furthermore, butin recovered ATP levels and succinate dehydrogenase activity that had been decreased by H2O2 treatment. We conclude these results suggest butin decreased mitochondrial ROS accumulation, balanced intracellular Ca2+ levels, and improved mitochondrial energy production, thus recovering mitochondrial function.