Direct therapeutic gene transfer is a promising tool to treat articular cartilage defects. Here, we tested the ability of an recombinant adeno-associated virus (rAAV) insulin-like growth factor I (IGF-I) vector to improve the early repair of cartilage lesions in vivo. The vector was administered for 3 weeks in osteochondral defects created in the knee joints of rabbits compared with control (lacZ) treatment and in cells that participate in the repair processes (mesenchymal stem cells, chondrocytes). Efficient IGF-I expression was observed in the treated lesions and in isolated cells in vitro. rAAV-mediated IGF-I overexpression was capable of stimulating the biologic activities (proliferation, matrix synthesis) both in vitro and in vivo. IGF-I treatment in vivo was well tolerated, revealing significant improvements of the repair capabilities of the entire osteochondral unit. IGF-I overexpression delayed terminal differentiation and hypertrophy in the newly formed cartilage, possibly due to contrasting effects upon the osteogenic expression of RUNX2 and β-catenin and to stimulating effects of this factor on the parathyroid hormone/parathyroid hormone-related protein pathway in this area. Production of IGF-I improved the reconstitution of the subchondral bone layer in the defects, showing increased RUNX2 expression levels in this zone. These findings show the potential of directly applying therapeutic rAAVs to treat cartilage lesions.