Trimetazidine (TMZ), as a metabolic regulator, has been widely testified to exhibit positive therapeutic effects on various disease models, but its role in diabetic retinopathy has not been reported. Therefore, this study was designed with the purpose of exploring the effects of TMZ on high-glucose (HG)-induced retinal endothelial dysfunction and its underlying mechanism. To establish DR model in vitro, 30 mM glucose was applied to induce human retinal endothelial cells (HRECs). Cell proliferation, invasion, and migration were examined by means of Cell Counting Kit-8, transwell, and wound healing assays, respectively. The tubule formation experiment was used to test the tubulogenesis ability and fluorescein isothiocyanate (FITC)-albumin was utilized to measure the permeability of monolayer HRECs. In addition, immunofluorescence and Western blot were employed to detect protein expression. Compared with the HG-induced group, TMZ concentration dependently inhibited the proliferation, migration, and angiogenesis of HG-induced HRECs, decreased the permeability of monolayer HRECs, and increased the protein expression levels of Claudin-5 and VE-cadherin. In addition, TMZ intervention increased the expression of p-PI3K, p-AKT, and p-mTOR but decreased the expression of LC3I, LC3II, and Beclin 1, which were then partially reversed by P13 K inhibitor (LY294002). Moreover, the autophagy agonist rapamycin (RAPA) was also testified to reverse the inhibitory effects of TMZ on the proliferation, migration, and angiogenesis of HG-induced HRECs. In summary, TMZ inhibited excessive autophagy by activating PI3K/Akt/mTOR pathway, thereby improving retinal endothelial dysfunction induced by HG.