Purpose: The purpose of this study is to examine in vivo osteogenesis of cultured human periosteal-derived cells and polydioxanone/ pluronic F127 scaffold. Methods: Two one-year-old miniature pigs were used in this study. 2×106 periosteal-derived cells in 1 mL medium were seeded by dropping the cell suspension into the polydioxanone/pluronic F127 scaffold. These cell-scaffold constructs were cultured in osteogenic Dulbecco`s modified Eagle`s medium for 7 days. Under general anesthesia with azaperone and tiletamine- zolazepam, the mandibular body and ramus of the pigs were exposed, Three bony defects were created, Polydioxanone/pluronic F127 scaffold with periosteal-derived cells and the scaffold only were implanted into each defect. Another defect was left empty. Twelve weeks after implantation, the animals were sacrificed. Results: New bone formation was clearly observed in the polydioxanone/pluronic F127 scaffold with periosteal-derived cells. Newly generated bone was also observed in the scaffold without periosteal-derived osteoblasts and empty defect, but was mostly limited to the periphery. Conclusion: These results suggest that cultured human periosteal-derived cells have good osteogenic capacity in a polydioxanone/ pluronic F127 scaffold, which provides a proper environment for the osteoblastic differentiation of these cells.