Diabetic retinopathy (DR) is highly related to blindness. Fortunately, Ginsenoside Rg1 (Rg1) can ameliorate retinal endothelial dysfunction, and this study thus aims to investigate its underlying mechanism under high glucose (HG) conditions. Human retinal microvascular endothelial cells (HRMECs) were exposed to HG, followed by 10 μM Rg1 treatment and cell viability detection. Protein expressions of LC3II/I and P62 as well as autophagosome formation were examined using Western blot and transmission electron microscopy, respectively, to unveil the effect of Rg1 on the autophagy of HG-exposed cells. Whether the ACE2/ Ang1-7/Mas axis participated in the Rg1-caused autophagy abatement as well as its upstream regulators were verified through treatment with Ang1-7, A779 or LY294002. Rg1 or Ang1-7 treatment enhanced cell viability, activated the ACE2/Ang1-7/Mas axis, downregulated LC3II/I, upregulated P62, and repressed autophagosome formation in HG-exposed HRMECs. However, the regulatory effects of Rg1 on cell viability, ACE2/Ang1-7/Mas axis and autophagy were reversed in the presence of either A779 or LY294002. Rg1 alleviates HG-induced autophagy of HRMECs through the ACE2/Ang1-7/Mas axis, mediated by the PI3K/Akt pathway, supporting the protective effect of Rg1 on retinal endothelium.