An extracellular matrix (ECM) utilized as a biomaterial can be obtained from organs of living organisms. Therefore, it has some limitations in its supply because of insufficient organs. Furthermore, therapeutic efficacy of ECMs varies depending on factors such as donor's health condition and age. For this reason, ECMs obtained from a cell line could be a good alternative because they can be produced under a controlled environment with uniform quality. Thus, the purpose of this study was to investigate the potential of the MC3T3-E1 cell line-derived ECM as bone graft. The optimized decellularization process was developed to separate the ECM from MC3T3-E1, osteoblast cell line, using Trypsin-EDTA and Triton X-100. The decellularized ECM was partially digested using pepsin. Also, human bone marrow-derived mesenchymal stem cells induced faster osteogenesis on the ECM-coated surface than on the collagen-coated surface. Partially digested ECM fragments were embedded on the polyethylene glycol scaffold without additional chemical modification or crosslinking. Micro-computed tomography and histological analysis results showed that the ECM in the scaffold promoted actual bone regeneration after in vivo implantation to a mouse calvarial defect model. This study suggests that the bone-specific ECM derived from the cell line can replace the ECM from organs for application in tissue engineering and regenerative medicine.