Objective To investigate the role of microRNA (miR)-20a-5p regulating vascular endothelial growth factor (VEGF) pathway in mice with oxygen-induced retinopathy (OIR). Methods The experiment was divided into normal group, model group, hyperoxia control group, miR-20a-5p high expression group, with 24 mice in each group. In addition to the normal group, the rest of the mice were placed in the oxygen tank with ( 75. 00 ± 2. 00)% oxygen concentration from the 7th day after birth to establish the OIR mice model, and after 5 days of continuous hyperoxia environment, they were fed in normoxia, at 1 day before the end of hyperoxia environment, 1 μL phosphate buffered saline (PBS) 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 did nothing. After the end of hyperoxia, the normal group was kept in the air for normal feeding. The experiment was carried out after normal air feeding for more 5 days. The expressions of miR-20a-5p, VEGF, vascular endothelial growth factor receptor (VEGFR)-1 and VEGFR-2 were detected by real-time fluorescence quantitative PCR ( qRT-PCR); retinal vascular morphology was observed by retinal patch; hematoxylin eosin (HE) 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 the 17th day of life, the radial large blood vessels from the optic disc extended roundly and irregularly, there were a lot of new blood vessels, the structure and distribution of neovascularization were disordered and the capillary network was occluded; and compared with the model group and the hyperoxia control group, the miR-20a-5p high expression group had no obvious radial vascular circuitous, less irregular vascular expansion and less neovascularization. Compared with those in the normal group, the level of miR-20a-5p in retina in model group and hyperoxia control group was lower (P< 0. 05), and the number of nuclei in retinal vascular endothelium, VEGF protein positive area percentage, expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher (P< 0. 05); meanwhile, compared with those in the model group and the hyperoxia control group, the level of miR-20a-5p in retina in 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, expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher ( P< 0. 05). Conclusions Increasing miR-20a-5p can inhibit VEGF pathway and decrease retinal neovascularization in OIR mice, so as to protect the retina of OIR mice.