A novel decellularized bone matrix was established by the supercritical carbon dioxide (SCCO2) extraction technology. The most commonly employed surgical technique is bone grafting, to enhance bone regeneration in orthopaedic surgery. The current study investigated the role of SCCO2 -decellularized bone matrix (scDBM) as bio-scaffold seeded with allogeneic adipose-derived mesenchymal stem cells (ADSC) for bone regeneration. The scanning electron microscopy (SEM) was used to evaluate the bio-scaffold produced by seeding ADSC to scDBM. The bio-scaffold was used to treat the rat segmental femoral defect model in a non-union model to examine the callus formation. Post-surgery after 12 and 24 weeks the osteotomy gap closure and defect area were analyzed using histological analysis. The ability of new bone formation by scDBM was evaluated by the immunohistochemical expression of Ki-67, BMP-2 and osteocalcin. A dose-dependent ADSC was found to adhere definitely to the scDBM bioscaffold as evidenced by SEM. The X-ray and H&E staining revealed callus formation and accelerated bone formation was detected in scDBM seeded with 2x106 and 5x106 ASCs group. The expression of Ki-67, BMP-2, and osteocalcin was increased scDBM seeded with 5x106 ASCs group at 12 weeks after surgery, compared to other experimental groups. To conclude, scDBM is an outstanding bio-scaffold that boosted the attachment and recruitment of mesenchymal stem cells. scDBM seeded with ASCs augmented new bone formation.