To explore the radiosensitizing effects of enzalutamide (ENZ), an androgen receptor (AR) antagonist, on glioblastoma (GBM) in vitro and in vivo.Apoptosis assay and cell cycle assay were performed on U87MG, U138MG, and Ln229 cell lines after the treatment of ENZ. Combenefit study was performed by combining different concentrations of ENZ and doses of radiation treatment (RT) on U87MG and MGPP3 cell lines in vitro. The relationship between AR and DNA repair genes were studied in TCGA database. RNA-seq experiments were performed on U87MG cells after ENZ treatment at various time points. Syngeneic orthotopic GBM mouse model was used to compare the treatment effects and overall survivals in the vehicle only, ENZ only, RT only, and ENZ plus RT groups.ENZ induced apoptosis in U87MG and Ln229 cells and increased the G2/M cell cycle arrest in all the three human GBM cell lines. Synergistic effects were observed in both human and mouse GBM cell lines when combining ENZ and RT treatment. In TCGA database, the expression levels of most DNA repair genes have positive correlations with AR gene in GBM patients. The RNA-seq results demonstrated that gene expression levels of DNA repair enzymes decreased in U87MG cells after 48 hours of ENZ treatment. All the mice with GBM implanted in brain treated with ENZ plus RT (8 Gy x2) survived at the end of the experiment with 100% rate of complete response. The overall survival (OS) of combined treatment group is significantly better than ENZ only (∼40%) and RT only (∼50%) group. The synergistic effects of combining treatment are at least partly due to much enhanced depletion of cancer stem cells than drug only or RT only treatment.We demonstrated the radiosensitizing effects of ENZ on GBM both in vitro and in vivo indicating the potential clinical efficacy treating GBM patients with AR antagonist(s).N. Zhao: None. F. Wang: None. S. Wang: None. C. Lin: Research Grant; DHHS/NIH/NCI, The Otis Glebe Medical Research Foundation, BioMimetix JV, LLC. Leading departmental research activity; University of Nebraska Medical Center. C. Zhang: Research Grant; University of Nebraska Medical Center.