Elevated plasma levels of inorganic phosphate (Pi) are harmful, causing, among other complications, vascular calcification and defective insulin secretion. The underlying molecular mechanisms of these complications remain poorly understood. We demonstrated the role of Pi transport across the plasmalemma on Pi toxicity in INS-1E rat clonal β cells and rat pancreatic islet cells. Type III sodium-phosphate cotransporters (NaPi) are the predominant Pi transporters expressed in insulin-secreting cells. Transcript and protein levels of PiT-1 and -2, isotypes of type III NaPi, were up-regulated by high Pi incubation. In patch-clamp experiments, extracellular Pi elicited a Na(+)-dependent, inwardly rectifying current, which was markedly reduced under acidic extracellular conditions. Cellular uptake of Pi elicited cytosolic alkalinization; intriguingly, this pH change facilitated Pi transport into the mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, mitochondrial permeability transition, and endoplasmic reticulum stress-mediated translational attenuation, leading to reduced insulin content and impaired glucose-stimulated insulin secretion. Silencing of PiT-1/-2 prevented Pi-induced superoxide generation and mPT, and restored insulin secretion. We propose that Pi transport across the plasma membrane and consequent cytosolic alkalinization could be a therapeutic target for protection from Pi toxicity in insulin-secreting cells, as well as in other cell types.-Nguyen, T. T., Quan, X., Xu, S., Das, R., Cha, S.-K., Kong, I. D., Shong, M., Wollheim, C. B., Park, K.-S. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high phosphate-induced mitochondrial oxidative stress and defective insulin secretion.