Gene therapy is a promising strategy for bone regenerative medicine. Although viral vectors have been intensively studied for delivery of osteogenic factors, the immune response inevitably inhibits bone formation. Thus, safe and efficient non-viral gene delivery systems are in high demand. Toward this end, we developed a polyplex nanomicelle system composed of poly(ethyleneglycol) (PEG)-block-catiomer (PEG-b-P[Asp-(DET)]) and plasmid DNA (pDNA). This system showed little cytotoxicity and excellent transfection efficiency to primary cells. By the transfection of constitutively active form of activin receptor-like kinase 6 (caALK6) and runt-related transcription factor 2 (Runx2), the osteogenic differentiation was induced on mouse calvarial cells to a greater extent than when poly(ethylenimine) (PEI) or FuGENE6 were used; this result was due to low cytotoxicity and a sustained gene expression profile. After incorporation into the calcium phosphate cement scaffold, the polyplex nanomicelles were successfully released from the scaffold and transfected surrounding cells. Finally, this system was applied to in vivo gene transfer for a bone defect model in a mouse skull bone. By delivering caALK6 and Runx2 genes from nanomicelles incorporated into the scaffold, substantial bone formation covering the entire lower surface of the implant was induced with no sign of inflammation at 4 weeks. These results demonstrate the first success in in vivo gene transfer with therapeutic potential using polyplex nanomicelles.