The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1,25-dihydroxyvitamin D [1,25(OH)₂D₃] on magnesium transport. In this study, we examined the effect of 1,25(OH)₂D₃ on distal cellular magnesium uptake and the modulation of this response by extracellular Ca²⁺ and Mg²⁺ in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca²⁺ and Mg²⁺ concentrations to diminish peptide hormone-stimulated Mg²⁺ uptake. Mg²⁺ uptake rates were determined by microfluorescence in Mg²⁺-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)₂D₃ for 16-24 h stimulated basal Mg²⁺ uptake in a concentration-dependent manner from basal levels of 164 ± 5 to 210 ± 11 nM/s, representing a 28 ± 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)₂D₃-stimulated^.Mg²⁺ uptake (154 ± 18 nM/s), suggesting that 1,25(OH)₂D₃ stimulates Mg²⁺ uptake through gene activation and protein synthesis. Elevation of extracellular Ca²⁺ inhibited 1,25(OH)₂D₃-stimulated Mg²⁺ uptake (143 ± 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca²⁺ of 1,25(OH)₂D₃-stimulated Mg²⁺ uptake (202 ± 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca²⁺ on 1,25(OH)₂D₃-responsive Mg²⁺ entry. This showed that elevated extracellular calcium modulates 1,25(OH)₂D-mediated responses through the CaSR. In summary, 1,25(OH)₂D₃ stimulated Mg²⁺ uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca²⁺, acting via the CaSR, inhibited 1,25(OH)₂D₃-stimulated Mg²⁺ entry. These data indicate that 1,25(OH)₂D₃ has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.