Resistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Recently, the role of lysosome, especially lysosomal proteases, in radioresistance is being paid more and more attention to. Here, we investigated the radioresistant role of Cathepsin B (CTSB), one important member of cysteine proteases, in GBM cell lines. A protease array kit was used to test GBM cells before and after irradiation. Nude mice were implanted with GBM cells to generate orthotopic xenografts for in vivo studies. Response of U87 and U251 cells to treatment was examined using cell viability, flow cytometry. Cells were transfected with siRNA knockdown and gene expression constructs and molecules potentially mediating response were examined through western blot analysis, PCR and EdU assay. The results from protease array kit showed that CTSB was up-regulated the most among all proteases after irradiation. And this was verified by western blot analysis and immunohistochemistry of tumor samples both from in vivo study and clinical patients. Compared to negative control group, knocking down CTSB led to radiosensitivity. And this radiosensitive effect was achieved by decreasing homologous recombination (HR) efficiency. Further study showed that knocking down CTSB caused cell cycle arrested in G0/G1 phases, in which HR efficiency was impaired. Knocking down CTSB contributed to radiosensitivity in GBM cells by causing cell cycle arrest and down-regulating HR efficiency.