The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparin-like molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23. The present findings have revealed a novel MAPK-dependent mechanism of the regulation of phosphate uptake by FGF signaling.