PiT1 is a Na(+)-phosphate (P(i)) cotransporter located at the plasma membrane that enables P(i) entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P(i) to the cell. Moreover, the role of P(i) in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P(i) availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P(i) or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na(+)-P(i) transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P(i) transporter in cell proliferation, tumor growth, and cell signaling.