Objective To explore the mechanism of reactive oxygen species (ROS) generation on apoptosis of human osteoarthritic chondrocytes induced by cyclic stretch in vitro. Methods The human osteoarthritic chondrocytes were subjected to cyclic stretch at the frequency of 0.5 Hz with 20% elongation. The chondrocytes without cyclic stretch were used as a control. ROS generation in chondrocytes was inhibited by the antioxidant, N-acetyl-L-cysteine (NAC) and potentiated by the glutathione depleter, DL-buthionine-［S,R］-sulfoximine (BSO). Apoptosis was detected by flow cytometry. Intracellular ROS was detected using DCFH-DA and caspase-9 activity was measured using spectrophotometry. Results The cyclic stretch at the frequency of 0.5 Hz with 20% elongation induced ROS generation, and activation of caspase-9 and apoptosis in human osteoarthritic chondrocytes were significantly increased (P<0.05). The inhibition or potentiation of intracellular ROS by NAC or BSO could obviously inhibit or improve caspase-9 activity and apoptosis in chondrocytes under cyclic stretch (P<0.05). Conclusions Cyclic stretch-induced apoptosis in human osteoarthritic chondrocytes is mediated by ROS generation and activation of caspase-9. Suppression of ROS can prevent chondrocytes from apoptosis induced by cyclic stretch.