Magnesium is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the CLDN16-encoded tight junction protein, claudin-16. However, the role of 1,25-dihydroxyvitamin D [1,25(OH)2VitD] in renal Mg2+ handling is unclear. We have shown that Mg2+ depletion increases and 1,25(OH)2VitD inhibits CLDN16 transcription. We have now explored further the molecular mechanisms underlying the effect of 1,25(OH)2VitD on claudin-16-mediated Mg2+ transport. Adult mice received parenteral 1,25(OH)2VitD or 1,25(OH)2VitD combined with either high-Mg2+ or low-Mg2+ diets. Administration of 1,25(OH)2VitD enhanced urinary excretion of Mg2+ and Ca2+. The 1,25(OH)2VitD also increased renal Ca2+-sensing receptor (CaSR) mRNA and decreased renal claudin-16 and claudin-19 mRNA and claudin-16 protein, but did not affect renal claudin-2 mRNA. The 1,25(OH)2VitD reversed the expected increase in claudin-16 mRNA in Mg2+-depleted animals. Comparably treated HEK 293 cells showed similar changes in claudin-16 mRNA, but 1,25(OH)2VitD did not alter mRNA of the TRPM6 Mg2+ channel. A luciferase reporter vector containing 2.5 kb of 5′-flanking DNA sequence from human CLDN16 (hCLDN16) was transfected into HEK 293 and OK cells. The hCLDN16 promoter activity was modestly decreased by 1,25(OH)2VitD, but markedly inhibited in HEK 293 cells coexpressing CaSR. Coexpression in OK cells of dominant-negative CaSR completely abolished inhibition of hCLDN16 promoter activity by 1,25(OH)2VitD. The 1,25(OH)2VitD-induced decrease in hCLDN16 promoter activity was attenuated in Mg2+-depleted HEK 293 cells. In conclusion, 1,25(OH)2VitD transcriptionally inhibits claudin-16 expression by a mechanism sensitive to CaSR and Mg2+. This renal effect of 1,25(OH)2VitD may serve as an adaptive response to the 1,25(OH)2VitD-induced increase in intestinal Mg2+ absorption.