MYC is a pleiotropic transcription factor that coordinates the expression of diverse programs that are together necessary for the growth and expansion of somatic cells. The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) down-regulates MYC expression, but the exact mechanism is still elusive. We found in RWPE-1 normal human prostate cells that 1alpha,25-dihydroxyvitamin D(3) down-regulates MYC mRNA with a periodicity of 30-90 min. In silico screening of the MYC gene locus identified six putative binding sites [vitamin D response elements (VDREs)] for the vitamin D receptor (VDR). Two of these VDREs efficiently bound VDR-retinoid X receptor heterodimers in vitro, and their genomic regions associated with VDR in RWPE-1 cells. Gene-specific small inhibitory RNA silencing indicated that basal MYC mRNA expression, as well as its down-regulation, depended on the exchange factor TBL1X (transducer beta-like 1, X-linked), the corepressor silencing mediator for retinoid and thyroid hormone receptor, and histone deacetylases (HDACs) 2, 6, and 11. Assaying the association of these five proteins with the VDRE-containing genomic regions of the MYC gene locus showed characteristic ligand-dependent profiles of TBL1X, silencing mediator for retinoid and thyroid hormone receptor, HDAC6, and HDAC11, in particular on an evolutionarily conserved VDRE. In conclusion, our data suggest that dynamically composed protein complexes that dock via VDR to the two VDREs may explain the repression of the MYC gene.