Fibroblast growth factor (FGF)23 is proposed to play a physiological role in the regulation of phosphate and vitamin D metabolism; deranged circulatory levels of FGF23 cause several diseases with abnormal mineral metabolism. This paper presents a novel approach to analyze the mechanism of action of FGF23 using anti‐FGF23 monoclonal antibodies that can neutralize FGF23 activities both in vitro and in vivo. We developed two antibodies (FN1 and FC1) that recognize the N‐ and C‐terminal regions of FGF23, respectively. Both FN1 and FC1 inhibited FGF23 activity in a cell‐based Klotho‐dependent reporter assay. Their administration caused marked increases in serum phosphate and 1,25D levels in normal mice. These changes were accompanied by altered expression in the kidney of type IIa sodium‐phosphate cotransporter, 25‐hydroxyvitamin‐D‐1α‐hydroxylase, and 24‐hydroxylase. Thus, this study using neutralizing antibodies confirms that FGF23 is a physiological regulator of phosphate and vitamin D metabolism. We addressed the mechanism of action for these neutralizing antibodies. Structural analysis of the FGF23/FN1‐Fab complex showed that FN1 masked putative FGF receptor‐binding sites in the N‐terminal domain of FGF23, whereas biochemical analyses showed that FC1 interfered with the association between FGF23 and Klotho by binding to the C‐terminal domain of FGF23. Taken together, our results suggest that the N‐ and C‐terminal domains of FGF23 are responsible for association with cognate FGF receptors and Klotho, respectively, and that these interactions are indispensable for FGF23 activity.