Bone tissue engineering provides a substitute for bone transplantation to address various bone defects. However, bone regeneration involves a large number of cellular events. In addition, obtaining sufficient source material for autogenous bone or alloplastic bone substitutes remains an unsolved issue. In previous studies, it was confirmed that bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs) had the capacity to promote bone regeneration. Additionally, bone morphogenetic protein-2 (BMP-2) has been demonstrated to be an active inducer of osteoblast differentiation. Therefore, the aim of the present study was to produce an effective integration system, including a scaffold, reparative cells and growth factors, that may enhance bone regeneration. Firstly, bone marrow-derived BMSCs and EPCs were isolated and identified by flow cytometry. Cell proliferation ability, secreted BMP-2 levels and alkaline phosphatase (ALP) activity were highest in the cell sheets containing BMP-2-modified BMSCs and EPCs. In addition, the expression levels of osteogenesis-associated genes, including runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5), ALP and integrin-binding sialoprotein (Ibsp), and osteogenesis-associated proteins, including Runx2, Dlx, ALP, Ibsp, vascular endothelial growth factor, osteonectin, osteopontin and type I collagen, gradually increased during the co-culture of ad-BMP-2-BMSCs/EPCs. The levels of these genes and proteins were increased compared with those observed in the BMSC, EPC and BMP-2-modified BMSC groups. Finally, scanning electron microscopy observation also demonstrated that the BMP2-modified BMSCs were able to combine well with EPCs to construct a cell sheet for bone formation. Collectively, these results describe an adenovirus (ad)-BMP2-BMSCs/EPCs co-culture system that may significantly accelerate bone regeneration compared with a BMSCs/EPCs co-culture system or ad-BMP2-BMSCs alone.