Osteoarthritis (OA), one of the most common joint diseases with unknown etiology, is characterized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes. The purpose of this study is to elucidate the molecular mechanisms of H₂O₂-mediated rabbit chondrocytes apoptosis. CCK-8 assay showed that H₂O₂ treatment induced a remarkable reduction of cell viability, which was further verified by the remarkable phosphatidylserine externalization after H₂O₂ treatment for 1 h, the typical characteristics of apoptosis. H₂O₂ treatment induced a significant dysfunction of mitochondrial membrane potential (ΔΨm), but did not induce casapse- 9 activation, indicating that H₂O₂ treatment induced caspase-independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently prevented H₂O₂-induced apoptosis. H₂O₂ treatment induced a significant increase of caspase-8 and -3 activation, and inhibition of caspase-8 or -3 significantly prevented H₂O₂-induced apoptosis, suggesting that the extrinsic pathway played an important role. Collectively, our findings demonstrate that H₂O₂ induces apoptosis via both the casapse-8-mediated extrinsic and the caspase-independent intrinsic apoptosis pathways in rabbit chondrocytes.