Matrix metalloproteinases (MMPs) degrade extracellular matrix and regulate many functions including cell signaling. Oxidative stress is implicated in the development of diabetic retinopathy, and MMP-2, the most ubiquitous member of the MMP family, is sensitive to oxidative stress. This study aimed to determine the regulation of MMP-2 by oxidative stress in the development of diabetic retinopathy and the role of MMP-2 in the apoptosis of retinal capillary cells. The effects of mitochondrial superoxide scavenger on glucose-induced alterations in MMP-2, and its proenzyme activator MT1-MMP and physiological inhibitor TIMP-2, were determined in retinal endothelial cells, and the regulation of their glucose-induced accelerated apoptosis by the inhibitors of MMP-2 was accessed. To confirm in vitro results, the effects of antioxidant supplementation on MMP-2, MT1-MMP, and TIMP-2 were investigated in the retina of streptozotocin-induced diabetic rats. Glucose-induced activation of retinal capillary cell MMP-2 and MT1-MMP and decrease in TIMP-2 were inhibited by superoxide scavengers, and their accelerated apoptosis was prevented by the inhibitors of MMP-2. Antioxidant therapies, which have been shown to inhibit oxidative stress, capillary cell apoptosis, and retinopathy in diabetic rats, ameliorated alterations in retinal MMP-2 and its regulators. Thus, MMP-2 has a proapoptotic role in the loss of retinal capillary cells in diabetes, and the activation of MMP-2 is under the control of superoxide. This suggests a possible use of MMP-2-targeted therapy to inhibit the development of diabetic retinopathy.