PURPOSE: This study aimed to establish a high-fat diet (HFD)-induced rat model of type 2 diabetes mellitus (T2DM) and employed tandem mass tag (TMT) proteomics to search for novel interventional targets for nonproliferative diabetic retinopathy (NPDR). PATIENTS AND METHODS: Six-week-old male Sprague-Dawley rats were randomly divided into a T2DM group fed a HFD and a normal group (NOR group) fed normal chow. After 6 w, the T2DM group was confirmed to have impaired glucose tolerance and was intraperitoneally injected with a single small dose of streptozotocin (STZ, 30 mg/kg), and blood glucose levels were monitored. The HFD was maintained for another 6 w, and an Evans blue assay and a dark-adapted electroretinogram (ERG) were conducted. Rat retinas were collected for morphology analysis, TMT proteomics analysis, and Western blotting. The expression patterns of selected differentially expressed proteins (DEPs) were validated in rat retinas via Western blotting and in aqueous humor from NPDR patients via slot blotting. RESULTS: After the 12-w HFD and STZ injection, the rats presented typical symptoms of T2DM. The retinas of T2DM rats presented pathological features of NPDR, including compromised scotopic ERGs, thinning of retinal layers, increased apoptosis and vascular leakage in the retina. Proteomic analysis identified DEPs and revealed profound dyslipidemia in T2DM rat retinas. The significant upregulation of the FABP3, TINAGL1, and COL4A3 proteins was validated in the retinas of the rats by Western blotting and in the aqueous humor of the NPDR patients by slot blotting. CONCLUSION: In a rat model of HFD-induced T2DM that is consistent with the natural history and pathological features of NPDR, proteomics and bioinformatics analyses identified FABP3, TINAGL1, and COL4A3 as the 3 key upregulated proteins in retinas for the first time. These findings are supported by technical and clinical validations and provide novel targets for NPDR intervention.