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Retinal ischemia is a common feature associated with several ocular diseases, including diabetic retinopathy. In this study, we investigated the effect of a retinal ischemia and reperfusion (I/R) injury on protein levels via a quantitative shotgun strategy using stable isotope dimethyl labeling combined with LC-MS/MS analysis. Based on the relative quantitation data of 1088 proteins, 234 proteins showed a greater than 1.5-fold change following I/R injury, 194 of which were up-regulated and 40 were down-regulated. Gene ontology analysis revealed that after I/R injury, there was an increase in the metabolic-process related proteins but a decline in cell communication, system process and transport-related proteins. A ribosome protein network and a secreted protein network consisting of many protease inhibitors were identified among the up-regulated proteins, despite a suppression of the mammalian target of rapamycin (mTOR) pathway following the I/R injury. A synaptic-related protein network was found to be significantly down-regulated, implicating a functional reduction of neurons following a retinal I/R injury. Our results provide new systems-biology clues for the study of retinal ischemia.