The cystic fibrosis transmembrane conductance regulator (CFTR) is linked to cell apoptosis and abundantly expressed in brain tissue. Mitochondrial oxidative stress plays a key role in activating apoptotic pathway following cerebral ischemia reperfusion (IR) injury. Reduced glutathione (GSH) is exclusively synthesized in cytosol but distributed in mitochondria. In the present study, we investigated whether CFTR affected mitochondrial oxidative stress via regulating GSH and thereby protected neurons against apoptosis following cerebral IR. Brains were subjected to global IR by four-vessel occlusion and CFTR activator forskolin (FSK) was used in vivo. CFTR silence was performed in vitro for neurons by RNA interference. We found that FSK suppressed neuronal apoptosis whereas CFTR silence enhanced neuronal apoptosis. FSK prevented the elevations in reactive oxygen species (ROS) and caspase activities while FSK inhibited the reductions in complex I activity and mitochondrial GSH level following IR. FSK decreased mitochondrial oxidative stress partially and preserved mitochondrial function. On the contrary, CFTR silence exaggerated mitochondrial dysfunction. CFTR loss increased hydrogen peroxide (H2O2) level and decreased GSH level in mitochondria. Importantly, we showed that CFTR was located on mitochondrial membrane. GSH transport assay suggested that GSH decrease may be a consequence not a reason for mitochondrial oxidative stress mediated by CFTR disruption. Our results highlight the central role of CFTR in the pathogenesis of cerebral IR injury. CFTR regulates neuronal apoptosis following cerebral IR via mitochondrial oxidative stress-dependent pathway. The mechanism of CFTR-mediated mitochondrial oxidative stress needs further studies. CFTR activation protects brain tissue against IR-induced apoptosis and oxidative stress. CFTR disruption enhances H2O2-induced neuronal apoptosis and CFTR loss leads to mitochondrial oxidative stress. CFTR regulates IR-induced neuronal apoptosis via mitochondrial oxidative stress. CFTR may be a potential therapeutic target to cerebral IR damage.