High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X‐linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short‐term (24 h) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in 1‐month‐old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH)2 D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co‐receptor Klotho, transcription factor mRNAs for early growth response‐1 transcription factor (Egr‐1), and c‐fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p‐FGFR1, p‐FGFR3, KLOTHO, p‐ERK1/2, C‐FOS, and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. J. Cell. Physiol. 232: 610–616, 2017. © 2016 Wiley Periodicals, Inc. A schematic model for the role of HMWFGF2 in the regulation of Pi by FGF23 signaling. Overexpression of HMWFGF2 in osteoblast linage cells induced excess FGF23 in blood. Elevated FGF23 can bind to its co‐receptors Fgfr1c/Fgfr3c and Klotho on kidney to activate ERK1/2 signaling pathway in distal tubule, this signaling was transferred to Npt2a in proximal tubules, resulting in Pi wasting. Blockade of FGF23 with FGF23Ab can rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg mice. Activated ERK1/2 can lead to hypophosphatemia by suppression of Cyp27b1 and induction of Cyp24.