The objective of the present study is to investigate the addition effect of transforming growth factor (TGF)-β1 on the bone formation at a rabbit skull defect induced by autologous bone marrow (BM). Following application of gelatin microspheres containing TGF-β1, with or without BM cells to skull bone defects, bone formation at the defect was assessed by soft X-ray, dual energy X-ray absorptometry (DEXA), and histological examinations. After implantation for 6 weeks, gelatin microspheres containing 0.05 μg of TGF-β1 plus 106 of BM cells induced bone formation at the 6 mm diameter bone defect. The defect was histologically closed by newly formed bone tissue, whilst both gelatin microspheres containing 0.05 μg of TGF-β1, and 106 and 107 of BM cells were ineffective. A DEXA experiment revealed that combination of gelatin microspheres containing TGF-β1 with BM cells enhanced the bone mineral density at the skull defect to a significantly greater extent than other agents. These findings indicate that a combination of gelatin microspheres containing TGF-β1 enabled BM cells to enhance the osteoinductive ability, resulting in bone formation even at the cell number at which BM cells alone were ineffective.