PURPOSE: We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1alpha,25-dihydroxyvitamin D(3). EXPERIMENTAL DESIGN: Profiling, transcriptional, and proliferation assays were undertaken in 1alpha,25(OH)(2)D(3)-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21). RESULTS: Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G(1) of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor alpha-negative (n = 7) tumors. 1alpha,25(OH)(2)D(3) resistance in cancer cell lines was targeted by cotreatments with either 1alpha,25(OH)(2)D(3) or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D(3) compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells. CONCLUSIONS: Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1alpha,25(OH)(2)D(3) resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D(3) compounds and low doses of TSA.