CD44 plays an important role in cancer metastasis, chemotherapy, and radiation resistance. The present study investigated the relationship of CD44 expression and radioresistance, and the potential mechanisms of CD44 in radiosensitivity using prostate cancer (CaP) cell lines. CD44 was knocked down in three CaP cell lines (PC-3, PC-3M-luc, and LNCaP) using small interfering RNA (siRNA) and clonogenic survival fractions after single dose irradiation were compared before and after CD44 knocking down (KD). The effect of radiation on cell cycle distribution was examined by flow cytometry and the cell cycle-related protein levels of phospho-Chk1 and phospho-Chk2 were ascertained by Western blotting. The expression of the DNA double strand break (DSB) marker-γH2AX was also quantified by immunofluorescence staining. Our results indicate that the down-regulation of CD44 enhanced radiosensitivity in PC-3, PC-3M-luc, and LNCaP CaP cells, the sensitizing enhancement ratio for these cell lines was 2.3, 1.3, and 1.5, respectively and that the delay of DNA DSB repair in low CD44-expressing KD CaP cells correlated with ineffective cell cycle arrest and the delayed phosphorylation of Chk1 and Chk2. These findings suggest that CD44 may be a valuable biomarker and a predictor of radiosensitivity in CaP treatment.