The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest because it allows cell implantation through minimally invasive surgical procedures. Previously, we showed that poly(lactic-co-glycolic acid) (PLGA) microspheres can be used as an injectable scaffold to engineer cartilage in the subcutaneous space of athymic mice. The purpose of this study was to determine whether PLGA microspheres can be used as an injectable scaffold to regenerate hyaline cartilage in the osteochondral defects of rabbit knees. A full-thickness wound to the patellar groove of the articular cartilage was made in the knees of rabbits. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into these osteochondral wounds. Both chondrocyte transplantations without PLGA microspheres and culture medium injections without chondrocytes served as controls. Sixteen weeks after implantation, chondrocytes implanted using the PLGA microspheres formed white cartilaginous tissues. Histological scores indicating the extent of the cartilaginous tissue repair and the absence of degenerative changes were significantly higher in the experimental group than in the control groups (P < 0.05). Histological analysis by a hematoxylin and eosin stain of the group transplanted with microspheres showed thicker and better-formed cartilage compared to the control groups. Alcian blue staining and Masson's trichrome staining indicated a higher content of the major extracellular matrices of cartilage, sulfated glycosaminoglycans and collagen in the group transplanted with microspheres than in the control groups. In addition, immunohistochemical analysis showed a higher content of collagen type II, the major collagen type in cartilage, in the microsphere transplanted group compared to the control groups. In the group transplanted without microspheres, the wounds were repaired with fibro-cartilaginous tissues. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for cartilage regeneration in a rabbit model of osteochondral wound repair.