Reconstructing segmental bone defects after resection of malignant bone tumors is a long-standing clinical problem. Treatment of bone tumors such as osteosarcoma involves chemotherapy. These chemotherapeutic agents are potent inhibitors of cell division and these drugs may affect regeneration of bone from osteoprogenitor cells. It may be possible to reconstruct segmental bone defects by a tissue-engineering approach. The aim of this study was to investigate the use of mesenchymal stem cells (MSCs) in a fibrin glue carrier to enhance bone regeneration after chemotherapy. Bone marrow was harvested from young adult male rats of the Wistar strain; stem cells were isolated and expanded. Bone regeneration in normal and chemotherapy-treated rats was investigated in 1.5-mm rat femoral defects created by osteotomizing the femur and stabilizing the femoral fragments by external fixation. The osteotomy gap was left either unfilled, filled with fibrin glue alone, or filled with glue containing stem cells. Bone formation within the gap was determined by radiography, dual-energy X-ray absorptiometry, and histology. It was shown that MSCs encapsulated within fibrin glue could remain viable for up to 96 h in tissue culture. Chemotherapy significantly reduced bone formation in unfilled defects and defects filled only with fibrin glue. When MSCs were used in conjunction with fibrin glue, even in non-chemotherapy-treated rats bone formation in the gap was significantly increased. Using stem cells, the effects of chemotherapy on bone formation could be alleviated by bone formation in the gap similar to that seen in non-chemotherapy-treated animals with MSCs. These studies demonstrated that a tissue-engineering approach in patients undergoing chemotherapy may be beneficial for treating segmental bone defects after tumor resection.