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PURPOSE We aimed to uncover the role of microRNA-181 (miR-181) in the disease onset of diabetic retinopathy (DR) and its underlying mechanism. METHODS MiR-181 levels in plasma and aqueous humor samples of non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR) and healthy subjects were analyzed by microarray and quantitative real-time polymerase chain reaction (qRT-PCR). Proliferative and migrative capacities of human retinal endothelial cells (hRECs) regulated by miR-181 were assessed. The binding between miR-181 and kruppel-like factor 6 (KLF6) was verified by dual-luciferase reporter assay. RESULTS MiR-181 was upregulated in plasma and aqueous humor samples of NPDR and PDR patients. Overexpression of miR-181 stimulated hRECs to proliferate and migrate. KLF6 was the downstream gene binding miR-181, which was involved in the regulation of hRECs by miR-181. CONCLUSIONS MiR-181 is upregulated in plasma and aqueous humor of DR patients. It enhances proliferative and migratory potentials of retinal endothelial cells by targeting KLF6.