Background Glioblastoma multiforme (GBM) is the most deadly and common cerebral malignant tumors in adults. Radiotherapy is one of the major treatments for GBM patients. Further work is urgently required to discover the mechanisms conferring GBM radioresistance.Methods Reverse transcription-quantitative PCR was applied to detect the relative expression of FoxO3a in glioma cell lines after radiation. Lentivirus mediated FoxO3a knockdown cells were constructed and were confirmed by RT-qPCR and western blot. Plate clone formation assay and flow cytometry was used to determine the colony formation ability and cell apoptosis following radiotherapy, respectively. DNA damage and levels of apoptotic proteins were determined by comet assay and western blot.Results FoxO3a was significantly up-regulated in GBM cells in response to radiation. Knockdown of FoxO3a enhances the responsiveness of glioma cells to radiotherapy, the cell clonogenic formation and DNA repair process with elevated the percentage of apoptotic cells. Moreover, suppression of FoxO3a increased pro-apoptotic proteins Caspase 3, Caspase 7 and Bax levels after radiotherapy.Conclusions The study indicated that suppression of FoxO3a increased radiosensitivity in glioma cells, which might be a potential diagnostic and therapeutic target. Inhibition of FoxO3a expression enhances the responsiveness of glioma cells to radiotherapy, and its proliferation ability is weaker than that of normal Foxo3 cells