Amauroderma rugosum (AR) is commonly recognized as a medicinal fungus, often used as an alternative to Ganoderma lucidum. There is a scarcity of comprehensive and in-depth research on its bioactive polysaccharides and their associated biological activities. Herein, we isolated the polysaccharide fractions extracted from AR (ARPs) and investigated their primary structure and anti-angiogenic activities, given that various diseases are associated with excessive angiogenesis. Four polysaccharide fractions including ARP-0, ARP-1, ARP-2, and ARP-5 were heteropolysaccharides with different molecular weights, monosaccharide compositions, and micromorphologies, highlighting their varying bioactive profiles. Treatment of human umbilical vein endothelial cells with these polysaccharide fractions showed that only ARP-5 inhibited cell proliferation after vascular endothelial growth factor (VEGF) stimulation. Similarly, ARP-5 inhibited human umbilical vein endothelial cells migration, invasion, and tube formation upon VEGF (50 ng/mL) treatment. Moreover, compared with the insignificant effects of ARP-0, ARP-1, and ARP-2, ARP-5 impeded angiogenesis in zebrafish embryos. Additionally, ARP-5 downregulated the VEGF/VEGFR2 signaling pathway in a dose-dependent manner, suggesting that ARP-5 exerts its anti-angiogenic activities by blocking the VEGF/VEGFR2-mediated angiogenesis signaling pathway. Taken together, the study findings shed light on the primary structure and bioactivity of ARPs.