Mesenchymal stem cells (MSCs) hold great promise for bone regeneration. However, the power of mesenchymal stem cells has not been applied to structural bone allografts in clinical practice. This study designed a new strategy to enhance the efficiency of allografts for segmental bone regeneration. Isolated MSCs were cultured to form a cell sheet. The MSC sheet was then wrapped onto structural allografts. The assembled structures were cultured in vitro to evaluate the differentiation potential of MSC sheet. The assembled structures were implanted subcutaneously into nude mice as well as into the segmental radius defect of rabbits to investigate the efficiency of MSC sheets to repopulate allografts for bone repair. MSC sheets, upon assembling on bone grafts, showed similar differentiation properties to the in situ periosteum in vitro. After implantation the MSC sheets accelerated the repopulation of bone grafts in nude mice. Moreover, MSC sheets induced thicker cortical bone formation and more efficient graft-to-bone end fusion at the segmental bone defects in rabbits. This study thus presented a novel, more efficient, and practical strategy for large weight-bearing bone reconstruction by using MSC sheets to deliver large number of MSCs to repopulate the bone allografts.