Hormones and vitamins play integral roles in modulating transcriptional activity of members of the nuclear hormone receptor (NR) superfamily. The nuclear receptor corepressor protein (N-CoR) is essential for the transcriptional repression by unliganded NRs. In an attempt to isolate novel components of the hormone signaling pathway, we used a yeast two-hybrid screen and identified human ATP-utilizing chromatin assembly and remodeling factor 1 (hAcf1) as an N-CoR interacting protein. A previously unrecognized function of hAcf1 in the repression of euchromatic genes in mammalian cells was found: hAcf1 plays key roles in the hormone responsiveness and in the transcriptional repression of specific class II NR-regulated genes. First, hormone treatment causes a significant release of hAcf1 from its target gene promoters. Second, hAcf1 is crucial for stabilizing the endogenous vitamin D receptor-N-CoR repression complex and N-CoR itself, in the vitamin D3-regulated IGF binding protein 3 and receptor activator of nuclear factor-κB ligand gene promoters, respectively. Third, RNA interference-mediated reduction of hAcf1 or vitamin D3 treatment differentially affects the histone modification profile and the histone occupancy in these genes. Together, these results establish that hAcf1 has a critical role in the transcriptional repression of specific NR-regulated genes and indicate that hAcf1 release and histone H3 and H4 eviction are novel mechanisms in hormone-induced gene activation.