Inorganic phosphate (Pi) plays a critical role in cell signaling and energy metabolism. However, elevated Pi concentration is correlated with serious cardiovascular complications, of which the underlying mechanisms have not been clearly elucidated. We have demonstrated that the cellular and mitochondrial Pi uptake followed by reactive oxygen species (ROS) generation acts as a critical role in high Pi-induced vascular calcification in rat aortic smooth muscle cells. Type III Na⁺-Pi cotransporters (PiT-1/2), the predominant plasmalemmal Pi transporters expressed in vascular smooth muscle cells, were up-regulated by high Pi incubation, both in total protein levels and surface abundance. Treatment of high Pi activated ERK/mTOR signaling. Using patch clamp technique, we observed that Pi-induced a Na⁺-dependent inwardly rectifying current in aortic smooth muscle cells as well as in PiT-1 overexpressed HEK cells. Cellular uptake of Pi elicited cytosolic alkalinization which can facilitate Pi transport into mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, upregulation of ostegenic genes and calcific changes in aortic smooth muscle cells. Vascular calcification by high Pi was prevented by mitochondrial ROS scavenger. Inhibition of Pi transport by PiT-1/2 knockdown or pharmacologic blocking of mitochondrial Pi transport or inhibition of ERK/mTOR signaling also restored all these pathogenic changes by high Pi. Taken together, we propose that not only mitochondrial oxidative stress but also Pi transport across plasma and mitochondrial membranes could be the therapeutic targets for Pi-induced vascular calcification and cardiovascular morbidities.