New treatment approaches are needed for hormone refractory prostate cancer. Oncolytic adenoviruses are promising anti-cancer agents, and their efficacy can be improved by combining with conventional therapies such as ionizing radiation. The aim of this study was to determine the timing of oncolytic adenovirus treatment with regard to radiation and study the mechanisms of synergy in combination treatment. Prostate cancer cells were infected with oncolytic adenoviruses, irradiated and synergy mechanisms were assessed. In vivo models of combination treatment were tested. Radiation and oncolytic viruses were synergistic when viral infection was scheduled 24 hr after irradiation. Combination of oncolytic adenovirus with radiotherapy significantly increased antitumor efficacy in vivo compared to either agent alone. Microarray analysis showed dysregulated pathways including cell cycle, mTOR and antigen processing pathways. Functional analysis showed that adenoviral infection was accompanied with degradation of proteins involved in DNA break repair. Mre11 was degraded for subsequent inactivation of Chk2-Thr68 in combination treated cells, while gammaH2AX-Ser139 was elevated implicating the persistence of DNA double strand breaks. Increased autophagocytosis was seen in combination treated cells. Combination treatment did not increase apoptosis or virus replication. The results provide evidence of the antitumor efficacy of combining oncolytic adenoviruses with irradiation as a therapeutic strategy for the treatment of prostate cancer. Further, these findings propose a molecular mechanism that may be important in radiation induced cell death, autophagy and viral cytopathic effect.