Transient receptor potential vanilloid type 6 (TRPV6) (ECAC2, CaT1) is the major ion channel in intestinal epithelial cell membranes responsible for calcium entry. Its expression is actively regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this report, we identify mechanisms integral to the regulation of TRPV6 by 1,25-(OH)2D3. Based upon the hormonal responsiveness of a 7-kb TRPV6 promoter fragment in intestinal cell lines, we used a chromatin immunoprecipitation (ChIP) scanning method to search for possible vitamin D receptor (VDR) and retinoid X receptor (RXR) regulatory regions within the TRPV6 locus. VDR/RXR binding was broad, ranging from -1.2 to -5.5 kb relative to the start site of TRPV6 transcription. These results were consistent with an in silico analysis that revealed putative regulatory elements (VDREs) located at -1.2, -2.1, -3.5, -4.3, and -5.5 kb. Despite the ChIP analyses, only regions of the TRPV6 gene that contained putative elements at -2.1 and -4.3 kb transferred 1,25-(OH)2D3 response to a heterologous promoter. Further study revealed that each of these two active regions contained composite VDREs comprised of two separate regulatory elements. Mutagenesis of the VDREs within the -2.1- and -4.3-kb region and the VDRE at -1.2 kb abrogated all response to 1,25-(OH)2D3 when examined within the natural TRPV6 promoter. A final ChIP assay revealed that VDR/RXR heterodimer binding to the TRPV6 gene was accompanied by both the recruitment of steroid receptor coactivator 1 as well as a broad change in histone 4 acetylation. These studies identify a mechanism by which 1,25-(OH)2D3 regulates the expression of TRPV6 in human intestinal cells.