Npt2 encodes a renal-specific, brush-border membrane Na⁺-phosphate (P_i) cotransporter that is expressed in the proximal tubule where the bulk of filtered P_i is reabsorbed. Mice deficient in the Npt2 gene were generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of P_i homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal P_i reabsorption in humans. Homozygous mutants (Npt2^−/−) exhibit increased urinary P_i excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal P_i reabsorption. However, unlike HHRH patients, Npt2^−/− mice do not have rickets or osteomalacia. At weaning, Npt2^−/− mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype. Our findings demonstrate that Npt2 is a major regulator of P_i homeostasis and necessary for normal skeletal development.