In this study, a novel three-dimensional (3D) heterogeneous/bilayered scaffold was constructed to repair large defects in rabbit joints. The scaffold includes two distinct but integrated layers corresponding to the cartilage and bone components. The upper layer consists of gelatin, chondroitin sulphate and sodium hyaluronate (GCH), and the lower layer consists of gelatin and ceramic bovine bone (GCBB). The two form a 3D bilayered scaffold (GCH–GCBB), which mimics the natural osteochondral matrix for use as a scaffold for osteochondral tissue engineering. The purpose of this study was to evaluate the efficacy of this novel scaffold, combined with chondrocytes and bone marrow stem cells (BMSCs) to repair large defects in rabbit joints. Thirty-six large defects in rabbit femoral condyles were created; 12 defects were treated with the same scaffold combined with cells (group A); another 12 defects were treated with cell-free scaffolds (group B); the others were untreated (group C). At 6 and 12 weeks, in group A hyaline-like cartilage formation could be observed by histological examination; the newly formed cartilage, which stained for type II collagen, was detected by RT–PCR at high-level expression. Most of the GCBB was replaced by bone, while little remained in the underlying cartilage. At 36 weeks, GCBB was completely resorbed and a tidemark was observed in some areas. In contrast, groups B and C showed no cartilage formation but a great amount of fibrous tissue, with only a little bone formation. In summary, this study demonstrated that a novel scaffold, comprising a top layer of GCH, having mechanical properties comparable to native cartilage, and a bottom layer composed of GCBB, could be used to repair large osteochondral defects in joints. Copyright © 2012 John Wiley & Sons, Ltd.