1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] increases serum calcium (Ca²⁺) concentration in vivo, an action counteracted by activation of the calcium-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca²⁺ excretion. Little is known, of the role the CaSR plays in this response through its potentially direct actions in kidney, gut and bone independent of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)₂D₃ in mice with disruption of both the CaSR and PTH genes (C-P-) compared to mice with disruption of the PTH gene (C+P-) or wild type mice (C+P+). After intraperitoneal injection of 0.5 ng/g BW of 1,25(OH)₂D₃, peak calcemic responses were observed at 24 hrs in all three genotypes, in association with 1) greater increases in serum Ca²⁺ in C-P- mice than in the other genotypes on a Ca²⁺-replete diet that was attenuated by a Ca²⁺-deficient diet and pamidronate, 2) increased urinary Ca²⁺ to creatinine ratios in the C+P- and C+P+ mice but a lowered ratio in the C-P- mice on a Ca²⁺-replete diet, and (3) no increases in calcitonin (CT) secretion in the C+P+ and C+P- mice and small increases in the C-P- mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca²⁺ transport at baseline in duodenum and kidney, and injection of 1,25(OH)₂D₃ increased gene expression 8 hours later. However, the changes in the genes evaluated did not fully explain differences in serum Ca²⁺ seen among the genotypes. In conclusion, mice lacking the CaSR have increased sensitivity to the calcemic action of 1,25(OH)₂D₃ in the hypoparathyroid state. This is principally from enhanced 1,25(OH)₂D₃-mediated gut Ca²⁺ absorption and decreased renal Ca²⁺ excretion, without any differences in bone related release of Ca²⁺ or CT secretion among the three genotypes that could explain the differences in their calcemic responses.