A major limitation of exogenous vitamin D3 administration for the treatment of prostate cancer is the marginal, if any, clinical efficacy. We dissected the basis for the resistance to the vitamin D3 antitumor properties and specifically examined the effect of its major catabolic enzyme, CYP24A1, in prostate cancer. Local CYP24A1 expression levels and the effect of selective modulation were analyzed using tissue microarrays from needle core biopsy specimens and xenograft-bearing mouse models. CYP24A1 mRNA was elevated in malignant human prostate tissues compared to benign lesions. High CYP24A1 protein levels were seen in poorly differentiated and highly advanced stages of prostate cancer and correlated with parallel increase in the tumor proliferation rate. The use of CYP24A1 RNAi enhanced the cytostatic effects of vitamin D3 in human prostate cancer cells. Remarkably, subcutaneous and orthotopic xenografts of prostate cancer cells harboring CYP24A1 shRNA resulted in a drastic reduction in tumor volume when mice were subjected to vitamin D3 supplementation. CYP24A1 may be a predictive marker of vitamin D3 clinical efficacy in patients with advanced prostate cancer. For those with up-regulated CYP24A1, combination therapy with RNAi targeting CYP24A1 could be considered to improve clinical responsiveness to vitamin D3.