The physiological functions of vitamin D are mediated by its metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) activating the transcription factor vitamin D receptor (VDR). The chromatin-organizing protein CTCF bound to 14% of the 1,25(OH)₂D₃-sensitive chromatin regions. Interestingly, 1,25(OH)₂D₃affected the chromatin association of CTCF providing an additional mechanism for the epigenome-wide effects of the VDR ligand. The 1,25(OH)₂D₃-modulated transcriptome of THP-1 cells comprised 1284 genes, 77.5% of which responded only 24 h after stimulation. During the 1,25(OH)₂D₃ stimulation time course the proportion of down-regulated genes increased from 0% to 44.9% and the top-ranking physiological function of the respective genes shifted from anti-microbial response to connective tissue disorders. The integration of epigenomic and transcriptomic data identified 165 physiologically important 1,25(OH)₂D₃target genes, including HTT and NOD2, whose expression can be predicted primarily from epigenomic data of their genomic loci. Taken together, a large number of 1,25(OH)₂D₃-triggered epigenome-wide events precede and accompany the transcriptional activation of target genes of the nuclear hormone.