The effectiveness of stem-cell based therapy has been hampered by the limited availability of stem cell sources, immune rejection, and difficulties in clinical adoption and regulatory approval. These obstacles can be partially circumvented by using in situ tissue engineering that recruits the endogenous stem/progenitor cells and provides cues to direct stem cell phenotype. Here, decellularized bone scaffold is mechanically modified by coating of collagen (Col)/hydroxyapatite (HA) mixture with optimal ratio and loaded with chemokine stromal cell-derived factor-1α (SDF-1α), in which endogenous stem cell recruitment can be improved by chemokine and stem cell fate can be regulated by matrix elasticity of the scaffold. This study shows that mesenchymal stem cells (MSCs) osteogenesis in vitro was enhanced by matrix elasticity and SDF-1α, and endogenous MSCs recruitment in subcutaneous implantation of rat was increased by the release of SDF-1α from the scaffold, and bone regeneration in rabbit large bone defect model was significantly improved by matrix elasticity and SDF-1α. In short, this study provides a new insight for developing novel engineered cell-free bone substitutes by mechanical modification for tissue engineering and regenerative medicine.