Vascular permeability is regulated mainly by the endothelial barrier and controls vascular homeostasis, proper vessel development, and immune cell trafficking. Several molecules are involved in regulating endothelial barrier function. Roundabout 4 (Robo4) is a single-pass transmembrane protein that is specifically expressed in vascular endothelial cells. Robo4 is an important regulator of vascular leakage and angiogenesis, especially under pathological conditions. The role of Robo4 in preventing vascular leakage has been studied in various disease models, including animal models of retinopathy, tumors, diabetes, and endotoxemia. The involvement of Robo4 in vascular endothelial growth factor and inflammation-mediated signaling pathways has been well studied, and recent evidence suggests that Robo4 modulates endothelial barrier function via distinct mechanisms. In this review, we discuss the role of Robo4 in endothelial barrier function and the underlying molecular mechanisms.