Objective To develop an extracellular matrix (ECM)-derived, integrated biphasie scaffold, and to investigate the regeneration potential of the scaffold loaded with chondrogenically induced bonemarrow mesenchymal stem cells (BMSCs) in the repair of large, high-load-bearing osteochoudral defects in a canine model. Methods The biphasie scaffolds were fabricated using cartilage ECM-derived Scaffold (Cartilage layer) and acellular bone matrix (Bone layer) by phase separation technique. Articular high-load-bearing osteochondral defects (4.2 mm diameter×6 mm deep) were created in femoral condyles. The defects were treated with constructs of a biphasic scaffold with chondrogenically induced BMSCs (experimental group) or cell-free scaffolds (control group). The repaired defects were evaluated for gross morphology, histological, and micro-CT analysis at 3 and 6 months post-implantation. Results Statistical analysis demonstrated that the gross and histological grading scores of the experimental group were always higher than those of the control group at both time points and that the scores for the 6-month experimental group was significantly higher than 3--month experimental group, while there was no significantly difference in control group between two time points. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at both 3 and 6 months, with no significant difference between the experimental and control groups. Conclusion The ECM-derived, integrated biphasic scaffold combined with chondrogenically induced BMSCs has high potential in repair of large, high-load-bearing osteochondral defects.