Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis-resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response-1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na+/Pi) cotransporter PiT-1. Since the effects of an inhibitor of Na+/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2α and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi-induced phosphorylation of ERK1/2 in the cells where the expression of PiT-1 was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT-1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210–1221, 2010. © 2010 Wiley-Liss, Inc.