The maintenance of cellular phosphate(Pi)homeostasis is of great importance in living organisms.The SPX domain-containing protein 1(SPX1)proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status.The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants,however,remains to be identified,as Pi itself does not bind to the SPX domain.Here,we report the identification of the inositol pyrophosphate InsP8 as a signaling molecule that regulates Pi homeostasis in Atabidopsis.Polyacrylamide gel electrophoresis profiling of InsP8 revealed that InsP8 level positively cor-relates with cellular Pi concentration.We demonstrated that the homologs of diphosphoinositol pentaki-sphosphate kinase(PPIP5K),VIH1 and VIH2,function redundantly to synthesize InsP8,and that the vih1 vih2 double mutant overaccumulates Pi.SPX1 directly interacts with PHR1,the central regulator of Pi star-vation responses,to inhibit its function under Pi-replete conditions.However,this interaction is compro-mised in the vih 1 vih2 double mutant,resulting in the constitutive induction of Pi starvation-induced genes,indicating that plant cells cannot sense cellular Pi status without InsP8.Furthermore,we showed that InsP8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1.Collectively,our study suggests that InsP8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.