Abstract Purpose: Neoadjuvant castration improves response to radiotherapy of prostate cancer. Here, we determine whether castration therapy impairs nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSB) by downregulating Ku70 protein expression. Experimental Design: Twenty patients with locally advanced prostate cancer were enrolled, and 6 to 12 needle core biopsy specimens were taken from the prostate of each patient before treatment. Bilateral orchidectomy was conducted in eight patients and 12 patients were treated with a GnRH agonist. After castration, two to four similar biopsies were obtained, and the levels of Ku70 and γ-H2AX foci were determined by immunofluorescence in verified cancer tissues. Results: We observed that the androgen receptor binds directly to Ku70 in prostate tissue. We also found a reduction of the Ku70 protein levels in the cell nuclei in 12 of 14 patients (P < 0.001) after castration. The reduction in Ku70 expression correlated significantly with decreased serum prostate-specific antigen (PSA) levels after castration, suggesting that androgen receptor activity regulates Ku70 protein levels in prostate cancer tissue. Furthermore, a significant correlation between the reductions of Ku70 after castration versus changes induced of castration of γ-H2AX foci could be seen implicating a functional linkage of decreased Ku70 levels and impaired DNA repair. Conclusions: Castration therapy results in decreased levels of the Ku70 protein in prostate cancer cells. Because the Ku70 protein is essential for the NHEJ repair of DSBs and its downregulation impairs DNA repair, this offers a possible explanation for the increased radiosensitivity of prostate cancer cells following castration. Clin Cancer Res; 19(6); 1547–56. ©2013 AACR.