Lifespan is thought to be largely determined by the combined effects of genetics and environmental factors including stress and nutrients. The effects on individual lifespan by a large amount of nutrients, such as carbohydrates and amino acids, and a limited amount of nutrients, such as vitamins, are well-studied. However, molecular mechanism of cellular lifespan determination by vitamins is not elucidated. Here, we show that pyridoxal 5’-phosphate (PLP), a biologically active form of vitamin B6 (VB6) consisting of pyridoxine, pyridoxal, and pyridoxamine, is a key factor to regulate cellular lifespan of budding yeast. Through microarray analysis of yeast aging cells, we found that the expression of SNZ1 gene encoding PLP synthase was increased in senescent cells. Yeast cells synthesize PLP via the salvage pathway as well as de novo, and import extracellular VB6 by a plasma membrane VB6 transporter Tpn1p. Deletion of SNZ1 and TPN1 shortened yeast replicative lifespan, which is defined by the number of daughter cells that a mother cell can generate before dying. Simultaneous deletion of SNZ1 and TPN1 severely shortened the lifespan. Excess supplementation of pyridoxine to medium restored the lifespan of the Δsnz1 and Δtpn1 mutants to wild-type level. SNZ1 overexpression in the Δtpn1 cells and TPN1 overexpression in the Δsnz1 cells prolonged the lifespan to normal. These data strongly suggested that VB6 regulates yeast replicative lifespan. To confirm this, we measured the intracellular content of VB6 by a microbiological assay and that of PLP by an enzymatic assay in the wild-type and mutant strains. The contents of VB6 and PLP in Δtpn1 mutant were decreased. Pyridoxine supplementation to Δtpn1 mutant culture recovered the VB6 and PLP contents to wild-type level. Overexpression of SNZ1 in the Δtpn1 cells did not change the VB6 content but elevated the PLP content. These results reveal that the short lifespan of Δtpn1 cells is caused by reducing the PLP content. Unexpectedly, the VB6 and PLP contents of Δsnz1 mutant were comparable to that of wild-type strain. We found that the VB6 and PLP contents were increased in senescent cells of both wild-type and Δsnz1 mutant strains, suggesting that the elevated PLP level in old cells is due to increasing VB6 uptake by Tpn1p but not due to increasing de novo PLP synthesis by Snz1p. We propose that PLP is required for maintenance of cellular lifespan against aging.