Transgenic mice overexpressing FGF23 (R176Q) (F^Tg) exhibit biochemical (hypophosphatemia, phosphaturia, abnormal 1,25(OH)₂D₃ metabolism) and skeletal (rickets and osteomalacia) abnormalities attributable to FGF23 action. In vitro studies now implicate the aging-related factor Klotho in the signaling mechanism of FGF23. In this study, we used a mouse genetic approach to validate in vivo the pivotal role of Klotho in the metabolic and skeletal derangements associated with FGF23 (R176Q) overexpression. To this end, we crossed mice heterozygous for the hypomorphic Klotho allele (Kl^+/-) to F^Tg mice and obtained F^Tg transgenic mice homozygous for the Kl-hypomorphic allele (F^Tg/Kl^-/-). Mice were sacrificed on postnatal day 50 and serum and tissues were procured for analysis and comparison to F^Tg, WT, and Kl^-/- controls. From 4 weeks onward, F^Tg/Kl^-/- mice were clearly distinguishable from F^Tg mice and exhibited a striking phenotypic resemblance to the Kl^-/- controls. Serum analysis for calcium, phosphorus, PTH, 1,25(OH)₂D₃, and ALP activity confirmed the biochemical similarity between the F^Tg/Kl^-/- and Kl^-/- mice and their distinctness from the F^Tg controls. The characteristic skeletal changes associated with FGF23 (R176Q) overexpression were also dramatically reversed by the absence of Klotho. Hence, the wide, unmineralized growth plates and the osteomalacic abnormalities apparent in trabecular and cortical bone were completely reversed in the F^Tg/Kl^-/- mice. Nevertheless, independent actions of Klotho on bone were also apparent, suggesting that an intact Klotho protein may modulate bone function independent of FGF23. In summary, our findings substantiate in vivo the essential role of Klotho in the mechanism of action of FGF23 in view of the fact that Klotho ablation fully reverses the complete spectrum of biochemical and most of the skeletal alterations resulting from FGF23 overexpression.