The human 25-hydroxyvitamin D₃ (25(OH)D₃) 1α-hydroxylase, which is encoded by the CYP27B1 gene, catalyzes the metabolic activation of the 25(OH)D₃ into 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃), the most biologically potent vitamin D₃ metabolite. The most important regulator of CYP27B1 gene activity is 1α,25(OH)₂D₃ itself, which down-regulates the gene. The down-regulation of the CYP27B1 gene has been proposed to involve a negative vitamin D response element (nVDRE) that is located ∼500 bp upstream from transcription start site (TSS). In this study, we reveal the existence of two new VDR-binding regions in the distal promoter, 2.6 and 3.2 kb upstream from the TSS, that bind vitamin D receptor–retinoid X receptor complexes. Since the down regulation of the CYP27B1 gene is tissue- and cell-type selective, a comparative study was done for the new 1α,25(OH)₂D₃-responsive regions in HEK-293 human embryonic kidney and MCF-7 human breast cancer cells that reflect tissues that, respectively, are permissive and non-permissive to the phenomenon of 1α,25(OH)₂D₃-mediated down-regulation of this gene. We found significant differences in the composition of protein complexes associated with these CYP27B1 promoter regions in the different cell lines, some of which reflect the capability of transcriptional repression of the CYP27B1 gene in these different cells. In addition, chromatin architecture differed with respect to chromatin looping in the two cell lines, as the new distal regions were differentially connected with the proximal promoter. This data explains, in part, why the human CYP27B1 gene is repressed in HEK-293 but not in MCF-7 cells.