Fibroblast growth factor (FGF)23 is a phosphaturic hormone that decreases circulating 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and elicits hypophosphatemia, both of which contribute to rickets/osteomalacia. It has been shown recently that serum FGF23 increases after treatment with renal 1,25(OH)₂D₃ hormone, suggesting that 1,25(OH)₂D₃ negatively feedback controls its levels by inducing FGF23. To establish the tissue of origin and the molecular mechanism by which 1,25(OH)₂D₃ increases circulating FGF23, we administered 1,25(OH)₂D₃ to C57BL/6 mice. Within 24 h, these mice displayed a dramatic elevation in serum immunoreactive FGF23, and the expression of FGF23 mRNA in bone was significantly upregulated by 1,25(OH)₂D₃, but there was no effect in several other tissues. Furthermore, we treated rat UMR-106 osteoblast-like cells with 1,25(OH)₂D₃, and real-time PCR analysis revealed a dose- and time-dependent stimulation of FGF23 mRNA concentrations. The maximum increase in FGF23 mRNA was 1,024-fold at 10⁻⁷ M 1,25(OH)₂D₃ after 24-h treatment, but statistically significant differences were observed as early as 4 h after 1,25(OH)₂D₃ treatment. In addition, using cotreatment with actinomycin D or cycloheximide, we observed that 1,25(OH)₂D₃ regulation of FGF23 gene expression occurs at the transcriptional level, likely via the nuclear vitamin D receptor, and is dependent on synthesis of an intermediary transfactor. These results indicate that bone is a major site of FGF23 expression and source of circulating FGF23 after 1,25(OH)₂D₃ administration or physiological upregulation. Our data also establish FGF23 induction by 1,25(OH)₂D₃ in osteoblasts as a feedback loop between these two hormones that completes a kidney-intestine-bone axis that mediates phosphate homeostasis.