The jawbone periosteum, the easily accessible tissue responding to bone repair, has been overlooked in the recent development of cell therapy for jawbone defect reconstruction. Therefore, this study aimed to elucidate the in vitro and in vivo biological characteristics of jawbone periosteum‐derived cells (jb‐PDCs). For this purpose, we harvested the jb‐PDCs from 8‐week‐old C57BL/6 mice. The in vitro cultured jb‐PDCs (passages 1 and 3) contained skeletal stem/progenitor cells and exhibited clonogenicity and tri‐lineage differentiation capacity. When implanted in vivo, the jb‐PDCs (passage 3) showed evident ectopic bone formation after 4‐week subcutaneous implantation, and active contribution to repair the critical‐size jawbone defects in mice. Molecular profiling suggested that R‐spondin 3 was strongly associated with the superior in vitro and in vivo osteogenic potentials of jb‐PDCs. Overall, our study highlights the significance of comprehending the biological characteristics of the jawbone periosteum, which could pave the way for innovative cell‐based therapies for the reconstruction of jawbone defects.