Abstract Vascular calcification (VC) is a degenerative disease that contributes to cardiovascular morbidity and mortality. A negative relationship has been demonstrated between VC and calcium sensing receptor (CaSR) expression in the vasculature. Of interest, vitamin D response elements, which allow responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], are present in the promoters of the CaSR gene. We hypothesized that 1,25(OH)2D3, by modulating CaSR expression in vascular smooth muscle cells (VSMCs), might protect against VC. Human VSMCs were exposed to increasing concentrations of 1,25(OH)2D3 (0.01–10 nmol/L) in noncalcifying (1.8 mmol/L) or procalcifying Ca2+0 condition (5.0 mmol/L). Using quantitative RT-PCR and Western blotting we observed a significant increase in both CaSR mRNA and protein levels after exposure to 1.0 nmol/L 1,25(OH)2D3. This effect was associated with a maximal increase in CaSR expression at the cell surface after 48 hours of 1,25(OH)2D3 treatment, as assessed by flow cytometry. Down-regulation of the vitamin D receptor by small interfering RNA abolished these effects. In the procalcifying condition, 1.0 nmol/L 1,25(OH)2D3 blocked the Ca2+0-induced decrease in total and surface CaSR expression and protected against mineralization. Down-regulation of CaSR expression by CaSR small interfering RNA abolished this protective effect. 1,25(OH)2D3 concentrations of 0.5 and 5.0 nmol/L were also effective, but other (0.01, 0.1, and 10 nmol/L) concentrations did not modify CaSR expression and human VSMC mineralization. In conclusion, these findings suggest that nanomolar concentrations of 1,25(OH)2D3 induce a CaSR-dependent protection against VC. Both lower and higher concentrations are either ineffective or may even promote VC. Whether this also holds true in the clinical setting requires further study.