Calcium has recently been shown to regulate fibroblast growth factor 23 (FGF-23), a bone-derived phosphate and vitamin D-regulating hormone. To better understand the regulation of FGF-23 by calcium, phosphorus, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D], and PTH, we examined FGF-23 expression under basal conditions and in response to PTH, doxercalciferol, or high-calcium diet treatment in Gcm2^−/− and Cyp27b1^−/− mutant mice. Gcm2^−/− mice exhibited low serum PTH and 1,25(OH)₂D concentrations, hypocalcemia, and hyperphosphatemia, whereas Cyp27b1^−/− mice had high PTH, undetectable 1,25(OH)₂D, hypocalcemia, and hypophosphatemia. Serum FGF-23 levels were decreased in both mutant models. Doxercalciferol administration increased serum FGF-23 levels in both mutant models. PTH administration to Gcm2^−/− mice also increased serum FGF-23 levels, in association with an increase in both 1,25(OH)₂D and calcium concentrations. Multiple regression analysis of pooled data indicated that changes in FGF-23 were positively correlated with serum calcium and 1,25(OH)₂D but not related to changes in serum phosphate concentrations. A high-calcium diet also increased serum FGF-23 concentrations in Cyp27b1^−/− mice in the absence of 1,25(OH)₂D and in Gcm2^−/− mice with low PTH. The addition of calcium to the culture media also stimulated FGF-23 message expression in MC3T3-E1 osteoblasts. In addition, FGF-23 promoter activity in cultured osteoblasts was inhibited by the L-calcium-channel inhibitor nifedipine and stimulated by calcium ionophores. The effects of chronic low calcium to prevent 1,25(OH)₂D and PTH stimulation of FGF-23 in these mutant mouse models suggest that suppression of FGF-23 plays an important physiological adaptive response to hypocalcemia.