The role of miRNAs in the radiosensitivity of glioma cells and the underlying mechanism is still far from clear. In the present study, we detected six downregulated and seven upregulated miRNAs in the serum after radiotherapy compared with paired serum samples before radiotherapy via miRNA panel PCR. Among these, miR-17-5p was highly reduced (fold change = -4.21). Further, we validated the levels of miR-17-5p in all serum samples with qRT-PCR. In addition, statistical analysis suggested that a reduced miR-17-5P level was positively associated with advanced clinical stage of glioma, incidence of relapse, and tumor differentiation. Moreover, we provided evidence that irradiation markedly activated autophagy and decreased miR-17-5p in the glioma cell line. Further, we demonstrated that irradiation-induced autophagy activation was mediated by beclin-1, and downregulation of beclin-1 via siRNA significantly abolished the irradiation-activated autophagy. Interestingly, we demonstrated that miR-17-5p could directly target beclin-1 via luciferase gene reporter assay. Exotic expression of miRNA-17-5p decreased autophagy activity in vitro. In nude mice, miRNA-17-5p upregulation sensitized the xenograft tumor to irradiation and suppressed irradiation-induced autophagy. Finally, pharmacal inhibition of autophagy markedly enhanced the cytotoxicity of irradiation in RR-U87 cells.