We studied the effect of prolonged activation of mitogen-activated protein kinase (MAPK) signaling on 1,25 dihydroxyvitamin D (1,25(OH)2D3) action in the immortalized human prostate epithelial cell line RWPE1 and its Ki-Ras transformed clone RWPE2. 1,25(OH)2D3-treatment caused growth arrest and induced gene expression in both cell lines but the response was blunted in RWPE2 cells. Vitamin D receptor (VDR) levels were lower in RWPE2 cells but VDR over-expression did not increase vitamin-D-mediated gene transcription in either cell line. In contrast, MAPK inhibition restored normal vitamin D transcriptional responses in RWPE2 cells and MAPK activation with constitutively active MEK1R4F reduced vitamin-D-regulated transcription in RWPE1 cells. 1,25(OH)2D3-mediated transcription depends upon the VDR and its heterodimeric partner the retinoid X receptor (RXR) so we studied whether changes in the VDR–RXR transcription complex occur in response to MAPK activation. Mutation of putative phosphorylation sites in the activation function 1 (AF-1) domain (S32A, T82A) of RXRα restored 1,25(OH)2D3-mediated transactivation in RWPE2 cells. Mammalian two-hybrid and co-immunoprecipitation assays revealed a vitamin-D-independent interaction between steroid receptor co-activator-1 (SRC-1) and RXRα that was reduced by MAPK activation and was restored in RWPE2 cells by mutating S32 and T82 in the RXRα AF-1 domain. Our data show that a common contributor to cancer development, prolonged activation of MAPK signaling, impairs 1,25(OH)2D3-mediated transcription in prostate epithelial cells. This is due in part to the phosphorylation of critical amino acids in the RXRα AF-1 domain and impaired co-activator recruitment. J. Cell. Physiol. 224: 433–442, 2010. © 2010 Wiley-Liss, Inc.