Radioresistance is responsible for treatment failure after radiotherapy in localized prostate cancer, while prostate cancer stem cells promote radioresistance by preferential activation of the DNA damage response. Chk1 inhibition has been shown to sensitize many tumor cells to radiation. However, whether Chk1 inhibition can potentiate the cytotoxic effects of radiation on prostate cancer stem cells remains to be elucidated. In this study, CD133+CD44+ cells were isolated using microbeads and were found to possess cancer stem cell properties. Using shRNA, Chk1 was knocked down in the sorted CD133+CD44+ cells. Our results demonstrated that Chk1 knockdown abrogated the radiation-induced G2/M arrest, inhibited DNA damage repair and promoted premature mitosis, leading to increased apoptosis in the radiated sorted CD133+CD44+ cells. Moreover, these effects were accompanied by caspase-2 activation and the inactivation of phosphorylated Cdc25C and Cdc2. Our results suggest that Chk1 knockdown increases the radiosensitivity of CD133+CD44+ prostate cancer stem cells. Chk1 knockdown in prostate cancer stem cells may be an effective therapeutic strategy against prostate cancer.