Angiogenesis is fundamental for the development of various pathological and physiological processes and can be modulated by the Slit-Robo signaling pathway. Three Slit proteins (Slit 1-3) and four Robo receptors (Robo 1-4) have been identified in mammals. Well known for their role in axon guidance, Slits and Robos are under dispute whether they are pro- or anti- angiogenic. This controversy necessitates deeper understanding regarding mechanisms that modulate Slit-Robo activities in angiogenesis. Our previous findings revealed that Slit3-induced angiogenesis is mediated by Robo4 and requires heparan sulfate, a type of glycosaminoglycan which is covalently linked to core proteins. However, how Slit3’s activities are tuned is yet to be discovered and the core protein(s) that mediates Slit3-induced angiogenesis is also undefined. Here, we describe two mechanisms that regulate the pro-angiogenic role of Slit3 based on our findings from immortalized diaphragmatic endothelial cells in vitro and matrigel plugs in vivo. Through investigation in ADAM-mediated ectodomain shedding, we identified a mechanism to generate soluble Robo4 which potently inhibits Slit3-induced angiogenesis. We also evaluated MMP2’s role as a regulator of Slit3-Robo4 signaling pathway. We verified that Glypican-4 binds to Robo4, mediates Slit3-induced angiogenesis and is subject to MMP2-mediated extracellular release. Therefore, this study is the first to identify a specific HSPG that mediates Slit3’s angiogenic activities and deepens our understanding regarding regulatory mechanisms of Slit3-induced angiogenesis.