The biological role of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] has generally been related to calcium homeostasis, but this hormone also has fundamental effects on processes of cellular proliferation and differentiation. The genomic actions of 1,25-(OH)₂D₃ are mediated by the vitamin D receptor (VDR) present in target cells. However, VDR transcriptional regulation is not well understood, probably due to the complexity of the VDR gene and its promoter. In the present study, it is demonstrated that administration of the pituitary transcription factor Pit-1 (originally found in the pituitary gland, but also present in other non-pituitary cell types and tissues) to the MCF-7 (human breast adenocarcinoma) cell line induces a significant increase in VDR mRNA and protein levels. Conversely, Pit-1-targeted siRNA markedly reduced expression of VDR in MCF-7 cells. Reporter gene assays demonstrated that the effect of Pit-1 is mediated by its binding to a region located between -254 and -246 base pairs from the VDR transcription start site. Selective mutations of this site completely abolished VDR transcription. ChIP analysis showed that binding of Pit-1 to the VDR promoter leads additionally to recruitment of CREB-binding protein, acetylated histone H4, and RNA polymerase II. Surprisingly, Pit-1 binding also recruits VDR protein to the VDR promoter. Using several cell lines with different levels of VDR expression, it was demonstrated that up-regulation of VDR transcription by Pit-1 is dependent on the presence of VDR protein, suggesting that transcriptional expression of VDR in a given cell type is dependent on, among other factors, its own expression levels.