LNCaP (androgen-sensitive human prostate cancer cells) and PC-3 cells (androgen-independent human prostate cancer cells) were used to investigate the anti-cancer effect of Ionizing Radiation (IR) combined with arsenic trioxide (ATO) and the underlying mechanisms in vitro and in vivo. We found that IR combined with ATO increases the therapeutic efficacy compared to individual treatments in LNCaP and PC-3 cells. Combined treatment induced autophagy and apoptosis in LNCaP cells, and mainly induced autophagy in PC-3 cells. The combined treatment induced cell death was mainly through inhibition of the Akt/mTOR signaling pathways. Furthermore, we found that the combined treatment of cells pre-treated with 3-methyladenine (3-MA) (an autophagy inhibitor) and LY294002 (a specific inhibitor of PI3K) resulted in a significant change in AO-positive cells and cytotoxicity. In in vivo study, the combination treatment possesses anti-tumor growth effect. These novel findings not only suggest a potential therapeutic strategy of the combined treatment for the treatment of androgen-dependent prostate cancer but also in androgen-independent prostate cancer.