A bilayered construct with or without adipose-derived stem cells (ASCs) was applied to repair full-thickness defects in the patellar groove of 18 rabbits. Non-treated and treated defects were divided into three groups: a control group (n = 12), a cell-free group (n = 12) and a cell-based group (n = 12). Histological appearance and grading were evaluated at 8 and 12 weeks. At 12 weeks, osteochondral-like tissues completely filled in the defects and integrated with host tissues in the cell-based group. The semi-quantitative score of the cell-based group (4.2 ± 1.2), which is a total score ranging from 0 (best) to 20 (worst), was significantly better than that of the other two groups (cell-free: 13.8 ± 2.5; control: 10.3 ± 2.4). This finding indicated that the bilayered constructs combined with ASCs could be an effective way to enhance osteochondral regeneration.