Retinopathy of prematurity (ROP) is an abnormal proliferative disease of retinal blood vessels. This study investigates the role of Robo4 in angiogenesis and its molecular mechanisms in the Oxygen-Induced Retinopathy (OIR) model and a hypoxic injury model of human umbilical vein endothelial cells (HUVECs). In the OIR model, Roundabout 4 (Robo4) expression was significantly decreased, while Robo4 overexpression inhibited neovascularization and alleviated retinal tissue damage. Under hypoxic injury-inducing conditions in HUVECs, Robo4 expression was inhibited, but overexpression enhanced cell proliferation and inhibited angiogenesis by negatively regulating the expression of adenosine diphosphate ribosylation factor 6 (ARF6) and vascular endothelial growth factor (VEGF). Molecular analysis revealed that Robo4 regulates VEGF expression through ARF6. Overexpression of Robo4 reduced ARF6 and VEGF expression, while knocking down Robo4 increased ARF6 and VEGF levels, confirming Robo4's role as a key regulator in the ARF6-VEGF axis. Additionally, Robo4 overexpression significantly inhibited the angiogenic capacity of HUVECs, and ARF6 overexpression partially reversed Robo4-mediated inhibition of angiogenesis. In the OIR model, Robo4 overexpression significantly suppressed pathological neovascularization, as evidenced by a significant reduction in the percentage of non-perfused areas. Furthermore, Robo4 overexpression downregulated ARF6 and VEGF expression levels and promoted the normalization and remodeling of retinal vascular structures. These findings suggest that Robo4 inhibits pathological angiogenesis by regulating ARF6 and VEGF, providing insights into its potential as a therapeutic target for retinal diseases like ROP.