Diabetic retinopathy, a retinal vascular disease, is inhibited in animals treated with aminoguanidine, an inhibitor of inducible nitric oxide synthase. This treatment also reduces retinal protein nitration, which is greater in diabetic rat retina than non-diabetic retina. As an approach to understanding the molecular mechanisms of diabetic retinopathy, we sought the identity of nitrotyrosine-containing proteins in retina from streptozotocin-induced diabetic rats and in rat retinal Muller cell line grown in high glucose (25 mM). Anti-nitrotyrosine immunoprecipitation products from rat retina and Müller cell were analyzed by LC MS/MS. Ten nitrated proteins in diabetic rat retina and 3 nitrated proteins in Müller cells grown in high glucose were identified; 3 additional nitrotyrosine-containing proteins were tentatively identified from diabetic retina. The identified nitrotyrosine-containing proteins participate in a variety of processes including glucose metabolism, signal transduction, and transcription/ translation. Among the nitrated proteins were insulin-responsive glucose transporter type 4 (GLUT-4) which has been previously implicated in the pathogenesis of diabetes mellitus, exocyst complex component Exo 70 which functions in insulin-stimulated glucose uptake of GLUT-4-containing vesicles, and fibroblast growth factor receptor 2 which influences retinal vascularization via fibroblast growth factor signaling. Nitration of tyrosine phosphorylation sites were identified in five proteins, including GLUT-4, exocyst complex component Exo70, protein tyrosine phosphatase eta (PTP-eta), sensory neuron synuclein, and inositol triphosphate receptor 3. Quantitation of nitration and phosphorylation at common tyrosine modification sites in GLUT-4 and PTP-eta from diabetic and nondiabetic animals suggests that nitration reduced tyrosine phosphorylation ~2x in these proteins from diabetic retina. The present results provide new insights regarding tyrosine nitration and its potential role in the molecular mechanisms of diabetic retinopathy.