Nuclear factor-kappaB provides an adaptive response to protect cancer cells against cytotoxicity induced by redox active therapeutics. RelB is uniquely expressed at a high level in prostate cancer with high Gleason scores. Recently, we showed that the level of RelB rapidly increases in androgen-independent prostate cancer cells after exposure to ionizing radiation (IR), leading to a reduction in intrinsic radiosensitivity. Here, we show that interaction of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] with the vitamin D receptor significantly enhances radiosensitivity of prostate cancer cells at clinically relevant radiation doses. The radiosensitization effect of 1alpha,25-(OH)(2)D(3) is mediated, at least in part, by selectively suppressing IR-mediated RelB activation, leading to a reduced expression of its target gene MnSOD, a primary antioxidant enzyme in mitochondria. These results suggest that suppression of manganese superoxide dismutase is a mechanism by which 1alpha,25-(OH)(2)D(3) exerts its radiosensitization effect and that 1alpha,25-(OH)(2)D(3) may serve as an effective pharmacologic agent for selectively sensitizing prostate cancer cells to IR via suppression of antioxidant responses in mitochondria.