The precise regulation of the body’s phosphate (Pi) homeostasis is a critical task. Treatment of hyperphosphatemia, which becomes inevitable in later stages of chronic kidney disease (CKD), is limited to dietary Pi restriction and oral Pi binders. Two transport proteins mediate renal Pi reabsorption, the sodium-phosphate cotransporters Npt2a and Npt2c. The former mediates the majority of renal Pi reabsorption (70-80%), which is a hormonally regulated process and requires parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). Currently, no renal Pi cotransporter is a pharmacological target. Studies were performed in vitro and in vivo employing a novel Npt2a inhibitor (Npt2a-I, PF-06869206). In opossum kidney (OK) cells, a model of the proximal tubule, the Npt2a-I caused a dose-dependent decrease of Na+-dependent Pi uptake (IC50: ~1.4 μmol/L). Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax. In vivo, the Npt2a-I induced a dose-dependent increase in urinary Pi excretion in wild-type mice (ED50: ~23 mg/kg), a finding completely absent in Npt2a-/- mice. The observed decrease in plasma Pi in WT mice is also absent in Npt2a-/- mice. Surprisingly the Npt2a-I-induced increase in urinary Na+ excretion was unaffected in Npt2a-/- mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a inhibitor on open probability of the epithelial Na+ channel (ENaC) in the cortical collecting duct. The effects on urinary Pi excretion and plasma Pi were also observed in a 5/6 nephrectomy (Nx) model but were somewhat attenuated. In addition, Sham and 5/6 Nx mice show a similar decrease in PTH in response to Npt2 inhibition. In summary, Npt2a inhibition is a possible treatment option in conditions where hyperphosphatemia is present, e.g., in severe CKD, acute tumor lysis syndrome, rhabdomyolysis, hemolysis, hyperthermia, profound catabolic stress, or acute leukemia.