We have evaluated the ectopic new bone formation effects of CPC (calcium phosphate cement) seeded with pBMP-2 (plasmids containing bone morphogenetic protein-2 gene) transfected canine bMSCs (bone marrow stromal cells) mediated by a non-viral PEI (polyethylenimine) derivative (GenEscort™ II) in nude mice. Canine bMSCs were transfected with pBMP-2 or pEGFP (plasmids containing enhanced green fluorescent protein gene) mediated by GenEscort™ II in vitro, and the osteoblastic differentiation was explored by ALP (alkaline phosphatase) staining, ARS (alizarin red S) staining and RT-qPCR (real-time quantitative PCR) analysis. Ectopic bone formation effects of CPC/pBMP-2 transfected bMSCs were evaluated and compared with CPC/pEGFP transfected bMSCs or CPC/untransfected bMSCs through histological, histomorphological and immunohistochemical analysis 8 and 12 weeks post-operation in nude mice. Transfection efficiency was up ∼35% as demonstrated by EGFP (enhanced green fluorescent protein) expression. ALP and ARS staining were stronger with pBMP-2 gene transfection, and mRNA expression of BMP-2 (bone morphogenetic protein-2), Col 1 (collagen 1) and OCN (osteocalcin) in pBMP-2 group was significantly up-regulated at 6 and 9 days. Significantly higher NBV (new bone volume) was achieved in pBMP-2 group than in the control groups at 8 and 12 weeks (P<0.05). In addition, immunohistochemical analysis indicated higher OCN expression in pBMP-2 group (P<0.01). We conclude that CPC seeded with pBMP-2 transfected bMSCs mediated by GenEscort™ II could enhance ectopic new bone formation in nude mice, suggesting that GenEscort™ II mediated pBMP-2 gene transfer is an effective non-viral method and CPC is a suitable scaffold for gene enhanced bone tissue engineering.