ABSTRACT Vitamin D receptor (VDR) is activated by natural ligands, 1α, 25-dihydroxy-vitamin D3 (1α, 25(OH)2-D3) and lithocholic acid (LCA). Our previous study shows that VDR is expressed in human hepatocytes, and VDR ligands inhibit bile acid synthesis and transcription of the gene encoding cholesterol 7α-hydroxylase (CYP7A1). Primary human hepatocytes were used to study LCA and 1α, 25(OH)2-D3 activation of VDR signaling. Confocal immunofluorescent microscopy imaging and immunoblot analysis showed that LCA and 1α, 25(OH)2-D3 induced intracellular translocation of VDR from the cytosol to the nucleus, and also plasma membrane where VDR co-localized with caveolin-1. VDR ligands induced tyrosine phosphorylation of c-Src and VDR, and their interaction. Inhibition of c-Src abrogated VDR ligand-dependent inhibition of CYP7A1 mRNA expression. Kinase assays showed that VDR ligands specifically activated the c-Raf/MEK1/2/ERK1/2 pathway, which stimulates serine phosphorylation of VDR and HNF4α, and their interaction. Mammalian two-hybrid assays showed a VDR ligand dependent interaction of nuclear receptor corepressor-1 (NCoR-1) and silencing mediator of retinoid and thyroid (SMRT) with VDR/RXRα. Chromatin immunoprecipitation assays revealed that an ERK1/2 inhibitor reversed VDR ligand-induced recruitment of VDR, RXRα and corepressors to human CYP7A1 promoter. In conclusion, VDR ligands activate membrane VDR signaling to activate the MEK1/2/ERK1/2 pathway, which stimulates nuclear VDR/RXRα recruitment of co-repressors to inhibit CYP7A1 gene transcription in human hepatocytes. This membrane VDR signaling pathway may be activated by bile acids to inhibit bile acid synthesis as a rapid response to protect hepatocytes from cholestatic liver injury.