Calcitriol (1α, 25(OH)(2)-Vitamin D3) binds to the vitamin D receptor (VDR) and regulates differentiation of the normal mammary gland, and may therefore be useful in breast cancer treatment or prevention. Many breast cancer cells are, however, resistant to Calcitriol. In this study, we investigated the resistance mechanism and the role of epigenetic silencing of VDR by promoter hypermethylation. Bisulfite sequencing of the VDR promoter region revealed methylated CpG islands at -700 base pairs (bp) upstream and near the transcription start site. VDR CpG islands were demethylated by 5'deoxy-azacytidine treatment, and this was accompanied by a parallel increase in VDR mRNA levels in breast cancer cell lines. Quantitative methylation-specific PCR analyses confirmed hypermethylation of these CpG islands in primary tumors, and its absence in normal breast tissue. VDR transcripts detected in breast cancers were predominantly 5'-truncated, while normal breast tissue expressed full-length transcripts. Consistent with this observation, genes containing the VDR-responsive element (VDRE), such as cytochrome p450 hydroxylases, p21 or C/EBP were underexpressed in breast cancers compared to normal breast samples. Expression of the active longer transcripts of VDR was restored with 5'deoxy-Azacytidine (AZA) treatment, with a concurrent increase in expression of VDRE-containing genes. Thus, promoter methylation-mediated silencing of expression of the functional variants of VDR may contribute to reduced expression of downstream effectors of the VDR pathway and subsequent Calcitriol insensitivity in breast cancer. These data suggest that pharmacological reversal of VDR methylation may re-establish breast cancer cell susceptibility to differentiation therapy using Calcitriol.