1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is widely recognized as a potent stimulator of intestinal calcium absorption, but a hormone that counterbalances its action (negative feedback) has been elusive. A bone-derived hormone, FGF-23, is hypothesized to be a long-awaiting answer of this question since 1,25(OH)2D3 enhances FGF-23 release from bone whereas FGF-23 reduces the renal synthesis of 1,25(OH)2D3. In the present study, male mice [Imprinting Control Region (ICR) strain; 7-week-old; n = 6-10 per group] were used and anesthetized by an intraperitoneal injection with 70 mg/kg sodium pentobarbitone. This study has been approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University. Here, we found that the mouse duodenal epithelial cells strongly expressed FGF receptor proteins as determined by immunohistochemistry, suggesting that these cells could directly respond to FGF-23. Intravenous administration of recombinant mouse FGF-23 significantly abolished the 1,25(OH)2D3-induced transcellular and paracellular calcium transport in the mouse duodenum, as determined by Ussing chamber technique and 45Ca radiotracer. However, FGF-23 did not affect the baseline duodenal calcium transport. At the molecular level, FGF-23 antagonized the 1,25(OH)2D3-induced increases in the mRNA levels of several duodenal calcium transporters, e.g., TRPV5, TRPV6, and calbindin-D9k, but not claudin-2 or -12. Although FGF-23 also reduced the effect of 1,25(OH)2D3 on the transepithelial water movement (a driving force for the solvent drag-induced calcium transport), it did not alter the paracellular permeability to 3H-mannitol or 14C-polyethylene glycol-4000. It could be concluded that FGF-23 was an efficient negative feedback regulator of the 1,25(OH)2D3-induced calcium transport in the mouse duodenum. Our findings have thus corroborated the existence of the bone-kidney-intestinal axis of the FGF-23/vitamin D system in the regulation of intestinal calcium absorption.