1α,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] increases serum Ca²⁺ concentration in vivo, an action counteracted by activation of the Ca²⁺-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca²⁺ excretion. Relatively little is known of the role the CaSR plays in this response through its potentially direct actions in kidney, gut, and bone independently of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)₂D₃ in mice with targeted disruption of both the CaSR and PTH genes (C⁻P⁻) compared with that in mice with disruption of the PTH gene alone (C⁺P⁻) or wild-type mice (C⁺P⁺). After intraperitoneal injection of 0.5 ng/g body wt 1,25(OH)₂D₃, peak calcemic responses were observed at 24 h in all three genotypes in association with 1) a greater increase 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 (UCa/Cr) 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 increase in calcitonin (CT) secretion in the C⁺P⁺ and C⁺P⁻ mice and a small increase in the C⁻P⁻ mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca²⁺ transport at baseline in the duodenum and kidney, and injection of 1,25(OH)₂D₃ increased gene expression 8 h later. However, the changes in the genes evaluated did not fully explain the differences in serum Ca²⁺ seen among the genotypes. In conclusion, mice lacking the full-length CaSR have increased sensitivity to the calcemic action of 1,25(OH)₂D₃ in the setting of PTH deficiency. 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.