Purpose: Cerebrovascular accidents are one of the most common and serious diseases in the adults. It is therefore important to determine the exact pathogenic mechanisms of ischemic brain injuries for development of proper treatment strategies. Materials and Methods: Levels of intracellular reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were determined using an inverted epifluorescence microscope and FACS analysis after staining with DCF-DA and TMRE, respectively. A middle cerebral artery occlusion model was utilized as an in vivo model of ischemic stroke. Results: Exogenous administration of hydrogen peroxide increased levels of intracellular ROS followed by hyperpolarization of ΔΨm. The increases of intracellular ROS and hyperpolarization of ΔΨm were significantly suppressed by inhibitors of mitochondrial electron transport chain complex I (rotenone and DPI). Pretreatment with rotenone exerted a neuroprotective effect from oxidative injuries in vitro and in vivo. Conclusion: These results collectively suggest that hyperactivation of mitochondrial respiratory function may be detrimental by paradoxical production of ROS in the clinical setting of ischemic brain injuries. These will provide us not only a fundamental understanding about pathogenesis of ischemic brain injuries but also a rationale for developing new neuroprotective therapies.