People have been looking for tissue engineering approaches to treat large segment bone defects as replacements of autologous bone method. Cell-seeded scaffolds are promising candidates, but lack of vascularization into the scaffolds has greatly hindered their applications. To address this problem, we used a co-culture of bone marrow derived mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs) on calcium phosphate ceramics scaffolds for better vascularization and thus osteogenesis. Different ratios of BMSC/EPC (3:1, 2:1, 1:1, 1:2, 1:3) in the co-culture were examined in vitro, and it was revealed that the optimal value for neovascularization and osteogenesis was 1:3 and 2:1, respectively. Then cell mixtures with the optimized ratios were cultured in distinct regions of volume-reduced scaffolds and allowed to culture for 7 days for sufficient cell adhesion and ingrowth, as demonstrated by cell proliferation throughout the scaffolds and enhanced expressions of cell markers and growth factors comparing to a mono-culture of BMSCs. Upon implantation into a rabbit large segmental bone defect model, the scaffolds with co-culture of the cells had better osteoid tissue formation and bone remodeling supported by neovascularization, comparing to scaffolds with mono-culture or without cells. Yet with sub-optimal efficacy comparing to autologous bone grafts, it was believed to be a promising candidate for treatment of large segment bone defects.