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As a basic member of the Class III histone deacetylases, SIRT1 has been implicated in the occurrence and progression of diabetic retinopathy (DR). The current study aimed to investigate the roles of SIRT1/miR-20a/Yse-associated protein (YAP)/hypoxia-inducible factor 1 α (HIF1α)/vascular endothelial growth factor A (VEGFA) in DR. The expression of SIRT1 was initially determined through quantitative RT-PCR and Western blot analysis following the successful establishment of a DR mouse model, followed by detection of SIRT1 catalytic activity. Retinal microvascular endothelial cells (RMECs) were cultured in media supplemented with normal glucose (NG) or high glucose (HG). Thereafter, SIRT1 was either silenced or overexpressed in RMECs, after which EdU staining and Matrigel-based tube formation assay were performed to assess cell proliferation and tube formation. The binding relationship between YAP, HIF1α, and VEGFA was further illustrated using dual-luciferase reporter assay. Preretinal neovascular cell number was tallied with the IB4-positive vascular endothelial cells, as determined by immunofluorescence. SIRT1 was poorly expressed in mice with DR and HG-treated RMECs with low catalytic activity. The proliferation and tube formation capabilities of RMECs were elevated under HG conditions, which could be reversed following overexpression of SIRT1. SIRT1 was identified as positively regulating the expression of miR-20a with YAP detected as the key target gene of miR-20a. Our data suggested that YAP could upregulate VEGFA via induction of HIF1α. Moreover, SIRT1 overexpression strongly repressed RMEC proliferation and angiogenesis, which could be reversed via restoration of YAP/HIF1α/VEGFA expression. Taken together, the key findings of our study suggest that upregulation of SIRT1 inhibits the development of DR via miR-20a-induced downregulation of YAP/HIF1α/VEGFA.