Lifespan of organisms is determined by genetic factors and environmental factors, such as stress and nutrients. Calorie restriction is well studied to extend organismal and cellular lifespan in a variety of eukaryotes from yeast to mammals. Phosphate, an essential nutrient for all organisms, has been reported to be involved in lifespan regulation: the mutant mice lacking klotho exhibits hyperphosphatemia to shorten lifespan. However, molecular mechanism of lifespan regulation by phosphate is not known. Here, we show that phosphate starvation response factors regulate replicative lifespan in budding yeast. Yeast replicative lifespan, the number of daughter cells produced by a mother cell, is usually measured on YPD medium, which contains high amounts of phosphate. To know the effect of extracellular phosphate on yeast lifespan, we measured replicative lifespan on synthetic high- and low-phosphate medium. Phosphate restriction did not extend the lifespan of wild-type strain. Deletion of PHO80 and PHO85, which encode cyclin and cyclin-dependent kinase in phosphate starvation response pathway, respectively, shortened the lifespan under both high and low phosphate conditions. Pho80p-Pho85p phosphorylates and exports transcription factor Pho4p from the nucleus under high phosphate condition, resulting in no expression of phosphate starvation response genes. Simultaneous deletion of PHO80 and PHO4 restored the lifespan to wild-type level, indicating highly expression of the Pho4p target genes by deletion of PHO80 causes short lifespan. We screened for Pho4p target gene mutations that recovered the lifespan of the Δpho80 cells, and found such mutations in VTC1, VTC2, and VTC4 genes. The VTC genes are transcriptionally regulated by Pho4p and are required for vacuolar polyphosphate accumulation. Deletion of PPN1, which encodes vacuolar polyphosphate phosphatase, also shortened the lifespan as did that of PHO80. In vivo 31P-nuclear magnetic resonance analysis showed that polyphosphate was accumulated in Δpho80 and Δppn1 mutants compared with the wild type, but not in Δpho80 Δpho4 mutant, indicating that accumulation of polyphosphate shortens yeast replicative lifespan.