Objective To investigate the role of microRNA ( miR) -20a-5p in the regulation of the vascular endothelial growth factor (VEGF) pathway in mice with oxygen-induced retinopathy ( OIR) . Methods Mice were divided into a normal group, model group, hyperoxia control group, and miR-20a-5p high expression group, with 24 mice in each group. With the exception of the normal group, the mice were placed in an oxygen tank with ( 75. 00 ± 2. 00) % oxygen concentration beginning postnatal day 7 to establish the OIR model, and after 5 days of continuous hyperoxia environment, they were returned to normal air conditions. At 1 day before the end of the hyperoxia environment, 1 μL phosphate buffered saline was injected into the vitreous cavity of the hyperoxia control group, 1 μL miR-20a-5p agomir ( 1 μmol / L) was injected into the vitreous cavity of the high expression group, and the model group received nothing. The normal group was kept in normal air conditions. The experiment was carried out after the mice were exposed to normal air for 5 more days. The expressions of miR-20a-5p, VEGF, VEGF receptor (VEGFR) - 1 and VEGFR-2 were detected by real-time fluorescence quantitative PCR, retinal vascular morphology was observed by retinal patch, hematoxylin and eosin staining was used to count the endothelial cells of retinal neovascularization, and VEGF-positive cells were detected by immunohistochemistry. Results In the model group and hyperoxia control group, on postnatal day 17, the large radial blood vessels extending from the optic disc were round and irregular. There were many new blood vessels; the structure and distribution of neovascularization were disordered and the capillary network was occluded. Compared with the model group and the hyperoxia control group, the miR-20a-5p high expression group had no obvious circuitous radial vasculature and had less irregular vascular expansion and neovascularization. Compared with those in the normal group, the retinal miR-20a-5p levels in the model group and hyperoxia control group were lower ( P< 0. 05) , and the number of nuclei in retinal vascular endothelium, VEGF protein-positive area percentage, and expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher ( P< 0. 05) . Compared with those in the model group and the hyperoxia control group, the retinal miR-20a-5p level in the miR-20a-5p high expression group was lower ( P< 0. 05 ) , and the number of nuclei in retinal vascular endothelium, VEGF protein-positive area percentage, and expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher ( P< 0. 05) . Conclusions Increasing miR-20a-5p inhibited the VEGF pathway and decreased retinal neovascularization in OIR mice, which may protect the retina of OIR mice.