1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] promotes intestinal absorption of calcium primarily by binding to the vitamin D receptor (VDR) and regulating gene expression. 1,25-(OH)₂D₃ also exerts rapid actions at the cell membrane which include increasing intracellular calcium levels and activating protein kinase cascades. To explore potential crosstalk between calcium signaling elicited by the nongenomic actions of 1,25-(OH)₂D₃ and the genomic pathway mediated by VDR, we examined the effects of activated Ca²⁺/calmodulin-dependent kinases (CaMKs) on 1,25-(OH)₂D₃/VDR-mediated transcription. Expression of a constitutively active form of CaMKIV dramatically stimulated 1,25-(OH)₂D₃-activated reporter gene expression in COS-7, HeLa, and ROS17/2.8 cell lines. Metabolic labeling studies indicated that CaMKIV increased VDR phosphorylation levels. In addition, CaMKIV increased the independent transcription activity of the VDR coactivator SRC1, and promoted ligand-dependent interaction between VDR and SRC coactivator proteins in mammalian two-hybrid studies. The functional consequences of this multi-faceted mechanism of CaMKIV action were revealed by reporter gene studies, which showed that CaMKIV and select SRC coactivators synergistically enhanced VDR-mediated transcription. These studies support a model in which CaMKIV signaling stimulates VDR-mediated transcription by increasing phosphorylation levels of VDR and enhancing autonomous SRC activity, resulting in higher 1,25-(OH)₂D₃-dependent interaction between VDR and SRC coactivators.