The reconstruction of bone defects remains a significant clinical problem. In this study, we constructed cell sheet from bone marrow stromal cells on normal culture plates by a simple method. The cell sheets showed evident mineralized nodules, high alkaline phosphatase activities, indicating their in vitro osteogenic potential. Then its osteogenic capability to heal critical-size rabbit calvarial defect was investigated. Forty adult New Zealand White rabbits were randomly divided into 4 groups of 10 animals each: (1) empty, (2) demineralized bone matrix (DBM) alone, (3) DBM/cell suspension, and (4) DBM/cell sheet. Specimens were harvested 6 and 12 weeks after implantation, respectively. Radiographic, histologic, and histomorphometric analyses were performed to evaluate the new bone formation inside the defect. The results revealed that the defect treated with DBM/cell sheet showed significantly more bone formation than other 3 groups (P < 0.05). Our study indicates that the cell sheet enhances bone regeneration in healing critical-size rabbit calvarial defect, and cell sheet-based engineered bone might be considered as potential substitutes for bone reconstruction.