Objective: This study aims to establish a T2DM rat model consistent with the natural history of the disease, and apply TMT proteomics technology to analyze the retina to reveal the pathogenic mechanism of NPDR and search for new targets for NPDR intervention. Methods: Six-week-old SD male rats were randomly divided into type 2 diabetes group (T2DM group) and normal group (NOR group). T2DM group rats were fed with high-fat diet containing 60% fat energy, while NOR group rats were fed with normal chow diet. After 6 weeks, oral glucose tolerance tests were conducted on the two groups of rats. Following confirmation of insulin resistance, the T2DM group rats were intraperitoneally injected with 2% STZ (30mg/kg), and blood glucose levels were monitored 72 hours later. The rats with random blood glucose levels higher than 16.7 mmol/L were fed with the high-fat diet for another 6 weeks, and then retinas were collected from the two groups of rats for TMT proteomic analysis. Results: After 72 hours and 6 weeks after STZ injection, the T2DM group rats showed typical symptoms of diabetes such as hyperglycemia, weight loss, increased food intake and water consumption, suggesting the establishment of a rat model of T2DM. The bioinformatics analysis results of proteomics reveal the close relationship between differentially expressed proteins, fatty acid metabolism, and angiogenesis. Conclusion: In a T2DM rat model consistent with the natural history of the disease, this study used TMT proteomics technology to deeply analyze the molecular mechanism of NPDR and revealed the key role of fatty acid metabolism in the pathogenesis of NPDR. In addition, Fabp3, Tinagl1, Col4a3, and Snrpd1, may subserve candidate targets for NPDR intervention.