〔Abstract〕 Objective Based on glycolysis of hypoxia inducible factor-1α(HIF-1α)/Bcl2/adenovirus E1B interacting protein 3(BNIP3) pathway, to study the mechanism of oxygen-induced retinal angiogenesis in neonatal mice. Methods Human umbilical vein endothelial cells(HUVECs) were divided into normoxic group, hypoxia+si-NC group, hypoxia +si-HIF-1α group and hypoxia+si-HIF-1α+BNIP group. In normoxic group, HUVECs were exposed to normoxic(21% O2) and cultured. Hypoxia +si-NC group, hypoxia +si-HIF-1α group and hypoxia +si-HIF-1α+BNIP3 group were treated with si-NC, si-HIF-1α or si-HIF-1α combined with BNIP3 plasmid for 36 h, and then exposed to hypoxia(1% O2) for culture. The autophagy, glycolysis, proliferation, migration and tube formation of mitochondria were investigated by immunofluorescence, metabolic measurement, cell viability, scratch experiment and tube formation experiment. On the 7th day after birth, C57BL/6J mice were randomly assigned to different treatment groups: control group, oxygen-induced retinopathy(OIR) group, OIR+si-HIF-1α group and OIR+si-BNIP group. The neovascularization and vascular occlusion were measured. Results Compared with normoxic group, the rate of LC3+MitoTracker+ spots, glucose uptake and lactic acid release in HUVECs in hypoxia +si-NC group increased significantly(P<0.001). Compared with hypoxia +si-NC group, the rate of LC3+MitoTracker+ spots, glucose uptake and lactic acid release in HUVECs in hypoxia +si-HIF-1α group decreased significantly(P<0.01). Compared with normoxic group, the proliferation activity of HUVECs in hypoxia +si-NC group decreased significantly(P<0.05), and the wound healing area and the number of tubes formed increased significantly(P<0.01). Compared with hypoxia+si-NC group, the proliferation activity of HUVECs in hypoxia +si-HIF-1α group decreased significantly at the 24th, 48th and 72th hours of culture(P<0.05), and the wound healing area and the number of tubes formed decreased significantly(P<0.001). Overexpression of BNIP3 reversed the effects of HIF-1α knock-down on mitochondrial autophagy, glycolysis and biological function. Compared with OIR group, the neovascularization and vascular occlusion areas in retina of mice in OIR+si-HIF-1α group and OIR+si-BNIP3 group reduced significantly(P<0.05). Conclusion HIF-1α/BNIP3 signaling pathway promotes mitochondrial autophagy activation in HUVECs under hypoxia, which plays an important role in controlling endothelial function and angiogenesis.