Plasma phosphate (P) levels are tightly controlled, and elevated plasma P levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of P reabsorption: Na-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of P reabsorption. The aim of the present study was to determine the in vitro effects of a novel Npt2a inhibitor (PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a) mice. In OK cells, Npt2a inhibitor caused dose-dependent reductions of Na-dependent P uptake (IC: ~1.4 μmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of P uptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-P media. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher for P in response to Npt2a inhibition with no significant change in apparent . Higher parathyroid hormone concentrations decreased P uptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor induced a dose-dependent increase in urinary P excretion in wild-type mice (ED: ~23 mg/kg), which was completely absent in Npt2a mice, alongside a lack of decrease in plasma P. Of note, the Npt2a inhibitor-induced dose-dependent increase in urinary Na excretion was still present 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 in the cortical collecting duct.