Passive paracellular proximal tubular (PT) and intestinal calcium (Ca²⁺) fluxes have been linked to active sodium (re)absorption. Although the epithelial sodium/proton exchanger, NHE3, mediates apical sodium entry at both these sites, its role in Ca²⁺ homeostasis remains unclear. We, therefore, set out to determine whether NHE3 is necessary for Ca²⁺ (re)absorption from these epithelia by comparing Ca²⁺ handling between wild-type and NHE3^−/− mice. Serum Ca²⁺ and plasma parathyroid hormone levels were not different between groups. However, NHE3^−/− mice had increased serum 1,25-dihydroxyvitamin D₃. The fractional excretion of Ca²⁺ was also elevated in NHE3^−/− mice. Paracellular Ca²⁺ flux across confluent monolayers of a PT cell culture model was increased by an osmotic gradient equivalent to that generated by NHE3 across the PT in vivo and by overexpression of NHE3. ⁴⁵Ca²⁺ uptake after oral gavage and flux studies in Ussing chambers across duodenum of wild-type and NHE3^−/− mice confirmed decreased Ca²⁺ absorption in NHE3^−/− mice compared with wild-type mice. Consistent with this, intestinal calbindin-D_9K, claudin-2, and claudin-15 mRNA expression was decreased. Microcomputed tomography analysis revealed a perturbation in bone mineralization. NHE3^−/− mice had both decreased cortical bone mineral density and trabecular bone mass. Our results demonstrate significant alterations of Ca²⁺ homeostasis in NHE3^−/− mice and provide a molecular link between Na⁺ and Ca²⁺ (re)absorption.