Malignant glioma is the most prevalent form of malignant brain tumor. Although radiotherapy is widely used in glioma treatment, the radioresistance of glioma cells limits the success of the glioma treatment. The lack of effective targets and signaling pathways to reverse glioma radioresistance is the critical obstacle in successful treatment. In this study, we demonstrate that mitochondrial ATP-sensitive potassium channels (mtK(ATP) channels) are overexpressed in glioma cells and are closely related to the malignancy grade and the overall survival of the patients. Importantly, we showed that mtK(ATP) channels could control glioma radioresistance by regulating reactive oxygen species (ROS)-induced ERK activation. The inhibition of mtK(ATP) channels suppresses glioma radioresistance by inhibiting ERK activation both in vitro and in vivo. These findings reveal the important roles of the mitochondria and mtK(ATP) channels as key regulators in the radioresistance of glioma cells, and suggest that mtK(ATP) channel blockers and MAPK/ERK kinase (MEK) inhibitors are potential targets for drug development of glioma treatments.