Disruption of the major renal Na/phosphate (Pi) cotransporter gene, Npt2a, in mice leads to a substantial decrease in renal brush border membrane Na/Pi cotransport, hypophosphatemia and appropriate adaptive increases in renal 25-hydroxyvitamin D₃- 1-hydroxylase (1OHase) activity and the serum concentration of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D). The latter is associated with increased intestinal Ca absorption, hypercalcemia, hypercalciuria and renal calcification in Npt2^-/- mice. To determine the contribution of elevated serum 1,25(OH)₂D levels to the development of hypercalciuria and nephrocalcinosis in Npt2-/- mice, we examined the effects of 1OHase gene ablation and long-term Pi supplementation on urine Ca excretion and renal calcification by micro-computed tomography. We show that the urine Ca/creatinine ratio is significantly decreased in Npt2^-/-/1OHase^-/- mice when compared to Npt2^-/- mice. In addition, renal calcification, determined by estimating the calcified volume to total renal volume (CT/TV), is reduced by ~80% in Npt2^-/-/1OHase^-/- mice when compared to that in Npt2^-/- mice. In Npt2^-/- mice derived from dams fed a 1% Pi diet and maintained on the same diet, we observed a significant decrease in urine Ca/creatinine that was also associated with ~80% reduction in CV/TV when compared with counterparts fed a 0.6% diet. Taken together, the present data demonstrate that both 1OHase gene ablation and Pi supplementation inhibit renal calcification in Npt2^-/- mice and that 1,25(OH)₂D is essential for the development of hypercalciuria and nephrocalcinosis in the mutant strain.