In this study, we surveyed the p-DNA-PKcs (Ser 2056) level in human glioma samples and observed that hyperactivation of DNA-PKcs was closely associated with both malignant progression and poor clinical outcome of glioma patients. We further explored the potential correlation between inhibition of DNA-PKcs and TMZ efficacy in glioma. The results demonstrate a striking synergistic effect between DNA-PKcs inhibitor KU0060648 and TMZ in glioma cells. Inhibition of DNA-PKcs enhances TMZ sensitivity mainly via suppression of AKT activation. This study provides a potential target for evaluating glioma progression and improving TMZ efficacy in glioma therapy.