To investigate the possible biological actions of 24,25‐dihydroxyvitamin D₃ (24,25(OH)₂D₃), a tibial fracture‐healing model was established in White Leghorn chicks. Three‐week‐old White Leghorn chicks fed a vitamin D₃–replete diet were divided into four groups (control, anesthetized, sham, and fractured). On varying days after tibial fracture (F) or sham manipulation (S), renal 25(OH)D₃‐1α‐hydroxylase and 25(OH)D₃‐24‐hydroxylase (24‐hydroxylase) activities and serum Ca²⁺ concentrations were measured. Metofane anesthesia was found to have no effect on the activity of either of the hydroxylases; the activities of the hydroxylases in the control, anesthetized, and sham‐operated birds were similar. By 10 days after tibial fracture, the renal 24‐hydroxylase activity increased more than 3‐fold in F (1.33 ± 0.07 pmol/mg of protein) as compared with S (0.42 ± 0.03 pmol/mg of protein) (p < 0.0001). A time‐dependent study of the renal 24‐hydroxylase activity during the fracture repair process revealed a slow increase from the first day after fracture, a higher activity at 8 days, which peaked at 10–11 days, which is consistent with the formation of the callus. The 24‐hydroxylase activity then returned to the same level as the sham group 14 days after fracture. There was no significant difference in serum Ca²⁺ levels between the F and S groups over the 3‐week postfracture period. Serum levels of vitamin D₃ metabolites were also measured during the fracture healing process: a 3.4× increase of the 24,25(OH)₂D₃ level in the fractured group (3.64 ± 1.16 nM) was observed as compared with the control groups (1.08 ± 0.49 nM) at 10 days after fracture (p = 0.068). No significant differences were observed in the plasma levels of 25(OH)D₃ or 1α,25(OH)₂D₃ between the group with a fracture and the controls. Exposure of primary chick kidney cells in culture to serum obtained from chicks with a tibial fracture for 20 h resulted in an ∼40% increase in the activity of the 24‐hydroxylase as compared with cells exposed to serum from control birds. These results suggest that 24,25(OH)₂D₃ is involved in the early process of fracture repair and that there is some form of physiological communication between the fractured bone and the kidney so as to increase the renal 24‐hydroxylase and the circulating concentration of this metabolite.