Diabetic retinopathy (DR) remains a leading cause of blindness in adults with diabetes mellitus. The pathogenesis of DR is a multifactorial process, and excessive oxidative stress and apoptosis in retinal endothelial cells (RECs) play vital roles. In this study, RECs were treated with 25mM glucose for three days, and we found that miR-145 was significantly downregulated in high glucose (HG)-treated RECs. Overexpression of miR-145 attenuated the HG-induced apoptosis, oxidative stress and inflammatory cytokines secretion in RECs. Besides, miR-145 overexpression also attenuated the increased TLR4 expression and NF-κB p65 nuclear translocation in HG-treated RECs. Luciferase reporter assay showed that miR-145 could specifically bind to the 3′UTR of TLR4, and overexpression of TLR4 abrogated the beneficial effects of miR-145 in HG-treated RECs. Therefore we suggested that miR-145 might be a potential therapeutic target for DR.