Retinal ischemia contributes to multiple ocular diseases while aminoguanidine (AMG) treatment significantly inhibits the neuronal and vascular degeneration due to acute retinal ischemia and reperfusion (I/R) injury. In the present study, two-dimensional differential in gel electrophoresis (2D DIGE) was applied to profile global protein expression changes due to retinal I/R injury, and the protection effects mediated by AMG. Retinal ischemia was induced by elevated intraocular pressure to 80–90 mmHg for 2 hours, and reperfusion was established afterward. Retinal tissues were collected 2 days after I/R injury. After 2D DIGE analysis, a total of 96 proteins were identified. Among them, 28 proteins were identified within gel spots whose intensities were normalized by AMG pre-treatment, pathway analysis indicated that most were involved in glycolysis and carbohydrate metabolism. Selected enzymes identified by MS/MS within these pathways, including transketolase, triosephosphate isomerase 1, aldolase C, total enolase, and pyruvate kinase were validated by quantitative Western blots. Glycolytic enzymes and other differentially regulated proteins likely play previously unrecognized roles in retinal degeneration after I/R injury, and inhibition of the resulting metabolic changes, using pharmacologically gents such as AMG, serve to inhibit the changes in metabolism and mitigate retinal degeneration. Select glycolytic enzymes may provide novel therapeutic targets for inhibiting the neuronal and vascular degeneration after retinal I/R injury.