The putative biological activity of 24,25-(OH)₂D remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following fracture in chicks. Our laboratory has generated a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25-(OH)₂D. We set out to study the role of CYP24A1 and 24,25-(OH)₂D in intramembranous bone formation during distraction osteogenesis in wild-type and Cyp24a1-deficient mice. Distraction osteogenesis was applied to mouse tibiae using a miniature external fixator apparatus. Histomorphometric parameters and gene expression differences between the mutant mice and the wild-type controls were measured using micro computed tomography and reverse-transcription quantitative PCR.There were no statistically significant differences between genotypes when bone volume/tissue volume ratios were calculated at mid distraction, end of distraction, mid consolidation, or end of consolidation. We measured reduced expression of the Col10a1 gene in the mutant vs. wild-type mice at mid distraction (0.4 ± 0.1 vs. 1.0 ± 0.2 respectively, p = 0.01). Similarly, we measured a significantly lower expression of the osteogenic marker Atf4 in mutant vs. wild-type mice at end of distraction (0.7 ± 0.1 vs. 1.0 ± 0.1 respectively, p = 0.01) and at mid consolidation (0.6 ± 0.1 vs. 1.0 ± 0.1 respectively, p = 0.0003). These results suggest moderate and restricted differences in chondrogenesis and osteogenesis that did not affect the volume of bone produced following distraction. We conclude that CYP24A1 activity is not essential for intramembranous bone formation.