Vitamin D anti-tumor effect is often found reduced in the late stages of cancer. To uncover vitamin D resistance mechanism, we established a vitamin D-resistant human prostate cancer LNCaP cell line, LNCaP-R, by chronic exposure of cells to 1α,25-dihydroxyvitamin D3 (1,25-VD). The vitamin D receptor (VDR)-mediated transcriptional activity was reduced in LNCaP-R, whereas VDR expression level and DNA-binding capacity were similar compared to parental cells (LNCaP-P). The expressions of the key factors involved in VDR transactivity, including CYP24A1 and VDR-associated proteins are all increased in LNCaP-R cells, and yet treatment with ketoconazole, P450 enzymes inhibitor, as well as trichostatin A (TSA), a histone deacetylase inhibitor, did not sensitize LNCaP-R cells response to vitamin D, suggesting that neither a local 1,25-VD availability, nor VDR-associated proteins are responsible for the vitamin D resistance. Interestingly, nuclear factor-kappaB (NF-κB) signaling, which is critical for 1,25-VD/VDR activity was found reduced in LNCaP-R cells, thereby treatment with NF-κB activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), can sensitize LNCaP-R vitamin D response. Together, we conclude that NF-κB signaling is critical for vitamin D sensitivity, and dysregulation of this pathway would result in vitamin D resistance and disease progression.