In this study, we reported that the p-DNA-PKcs (S2056) was significantly elevated in glioma compared with adjacent normal brain, and such a high level of phosphorylated DNA-PKcs tightly associated with malignancy of glioma, including clinical stage and the survival time of glioma patients, collectively demonstrating a pivotal role of DNA-PKcs in the progression of glioma. This study also investigated the role of DNA-PKcs in TMZ sensitivity in glioma cells. The results indicated that inhibition of DNA-PKcs activation decreased glioma cell malignancies and promoted TMZ efficacy. Specifically, we demonstrated that inhibitor of DNA-PKcs, namely the KU0060648, functioned mainly due to suppression of AKT signaling.