PURPOSE: The Roundabout (Robo) family of proteins is related to the transmembrane receptors and plays a major role in neurogenesis. However, the role of the Robo proteins in proliferative retinopathy has not yet been defined. This study was conducted to determine whether Robo1 is expressed in the retina of patients with proliferative retinal disease and whether it has a pathobiological role in the disease. METHODS: Immunohistochemistry was used to determine the presence and distribution of Robo1 in the pathologic membranes in proliferative retinopathy. Small interfering (si)RNA technology was used to knockdown Robo1 expression and to study its effects on retinal pigment epithelial (RPE) cells in vitro. The impact on PVR development of blocking Robo1 expression was determined by applying specific siRNA in a PVR rabbit model. The prevalences of PVR and retinal detachment were determined by indirect ophthalmoscope on days 1, 3, 7, 14, 21, and 28 after the injection of RPE cells into the vitreous. RESULTS: Immunohistochemistry showed that Robo1 expression was detected in GFAP-labeled glial cells and cytokeratin-labeled RPE cells in proliferative membranes. Robo1 expression was also detected in CD31-labeled vascular endothelial cells. Knockdown of Robo1 expression not only reduced human RPE cell proliferation in vitro but also effectively suppressed the development of PVR in a rabbit model. CONCLUSIONS: Robo1 is present in the extracellular matrix of proliferative membranes and may be derived from dedifferentiated RPE cells. Silencing the expression of Robo1 in RPE cells inhibited cell proliferation and suppressed the development of PVR in an animal model, indicating a potential therapeutic usefulness in treating PVR.