We evaluated a 5-year-old boy who presented with multiple fragility fractures of long bones. The proband had scoliosis, markedly thickened calvarium, craniosynostosis, and heterogeneity of long bone cortical thickness. Iliac crest biopsy showed increased bone turnover with increased osteoblast number, osteoid thickness, osteoclast number, and decreased bone matrix mineralization. Exome sequencing identified a de novo missense mutation in SP7 (c.926C>G:p.S309W) in the proband, which was not found in gnomAD and was predicted to be deleterious by multiple silico analyses. To test the impact of the SP7 variant, we infected mouse primary chondrocytes and mesenchymal stem cells with retrovirus expressing GFP, wild-type (WT) SP7, or the S309W SP7 variant, followed by induction of osteoblast differentiation. Compared with GFP, WT SP7 expectedly accelerated osteoblast differentiation and bone matrix mineralization, with an increase in alkaline phosphatase (ALP) and Alizarin Red staining. The S309W variant caused an aberrant expression profile. Real-time PCR showed elevated endogenous Sp7 and Col1a1 and decreased Sox9 and Col2a1, compared both to GFP and WT, suggesting accelerated osteoblast differentiation, but expression of Mmp13, Ibsp, and Bglap decreased, suggesting impaired bone matrix formation. Consistently, ALP and Alizarin Red staining was also decreased. Heterozygous knock-in mice with the S309W variant showed perinatal lethality, but a small number of mice were recovered. Micro-CT showed increased bone thickness in clavicles, ribs, and long bones. Trabecular bone density was decreased at the metaphyses but increased at the diaphyses in a heterozygous mouse. The marrow space was almost completely absent in the long bones in a mosaic heterozygous 20-wk mouse. A previously reported frameshift mutation in SP7 in humans caused recessive osteogenesis imperfecta, and biallelic knockout in mice caused lack of bone formation. In contrast, our patient showed a dominant phenotype, which differed markedly, with evidence of increased osteoblast numbers and heterogeneously increased bone formation but defective osteoblast function. Mice with this mutation also showed a dominant phenotype with increased bone formation. Taken together, the findings suggest that the SP7 variant in this patient is not a simple loss-of-function mutation causing a failure of osteoblast differentiation but instead causes more complex alterations in osteoblast differentiation.