Retinal neovascularization generally play roles in the formation of various severe eye diseases, such as age-related macular degeneration and diabetic retinopathy. The regulation of neovascularization-related pathways by Sirtuin 3 (Sirt3), a major mitochondrial NAD(+)-dependent deacetylase, give us a cue that Sirt3 may participate in the retinal neovascularization. However, the mechanism remains unclear. Here, we established a retinal neovascularization model by using human retinal endothelial cells (HRECs) under the induction of high glucose and insulin (HGI). With this model, Sirt3-expressing lentivirus was constructed and then used to investigate the effect of Sirt3 overexpression on the expression of migration-, neovascularization- and autophagy-related genes. After the treatment of HGI on HRECs, the mRNA and protein levels of migration-related genes, including matrix metalloproteinase-2 (MMP-2) and MMP-9, were significantly upregulated. Meanwhile, angiogenesis-related genes, including vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1 alpha (HIF-1 alpha), and insulin-like growth factor-1 (IGF-1) were promoted at both mRNA and protein levels. However, HGI had no clear effect on the mRNA and protein levels of microtubule associated protein 1 light chain 3 (LC3), an autophagy-related gene. When Sirt3 was overexpressed by lentivirus infection after HGI, the upregulation of MMP-2, MMP-9, VEGF, HIF-1 alpha, and IGF-1 were suppressed at both transcription and translation levels. At the same time, LC3 mRNA and LC3-II protein increased. These results suggest that Sirt3 may inhibit retinal neovascularization by regulating the migration-, neovascularization- and autophagy-related factors expression. Thus we argue that Sirt3 may be a potential candidate drug for curing various eye diseases induced by retinal neovascularization. (C) 2016 Elsevier Inc. All rights reserved.