Low blood concentrations of 25-hydroxyvitamin D(3) are associated with increased mortality, while some studies suggest improved cardiovascular outcomes with vitamin D(3) supplementation in chronic kidney disease. However, the physiological effects of vitamin D(3) on the cardiovascular system remain poorly understood making it difficult to determine whether vitamin D(3) supplementation might provide cardiovascular benefit or even cause harm. Thus here we investigated the effects of chronic 1,25-dihydroxyvitamin D(3) treatment on intracellular signaling in human coronary artery smooth muscle cells (HCASMCs) and found that 1,25-dihydroxyvitamin D(3) significantly potentiated endothelin (ET-1) signaling. Specifically, 1,25-dihydroxyvitamin D(3) (24-h pretreatment) caused a more than threefold enhancement in both ET-1-induced intracellular calcium mobilization and extracellular signal-regulated kinase (ERK) activation. This 1,25-dihydroxyvitamin D(3)-elicited signaling enhancement was not observed for either vasopressin or carbachol. With the use of endothelin receptor (ETR) isoform-selective antagonists, ETRA was found to be primarily responsible for the 1,25-dihydroxyvitamin D(3)-induced ET-1 responsiveness and yet ETRA mRNA expression and protein abundance were unaltered following 1,25-dihydroxyvitamin D(3) treatment. While there was an increase in ETRB mRNA expression in response to 1,25-dihydroxyvitamin D(3), the protein abundance of ETRB was again unchanged. Finally, ETRA/ETRB heterodimerization was not detected in HCASMCs in either the absence or presence of 1,25-dihydroxyvitamin D(3). Together, these data show for the first time that 1,25-dihydroxyvitamin D(3) enhances endothelin responsiveness in HCASMCs and that the effect is mediated through ETRA.