Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR.retinoid X receptor directly impacts both the distal and proximal regulatory domains.