•Sulfated mannoglucan, JCS1S2 could disrupt angiogenesis both in vitro and in vivo.•JCS1S2 bound to both VEGF and VEGFR2 to inactivate VEGFR2 phosphorylation.•JCS1S2 downregulated the expression of VEGF and its transcription factor AP-1.•JCS2S2 impeded angiogenesis via blocking VEGFR2/Erk/VEGF signaling. More and more evidences suggested that sulfated natural glycans had impact on angiogenesis. However, the molecular targets and functional mechanism of glycans are still vague. JCS1S2 was the sulfated mannoglucan featured with a backbone of 1, 4-linked β-Manp and 1, 4-linked α-Glcp with sulfation at C-6 of β-Manp and α-Glcp residues, respectively. The degree of substitution of this sulfated polysaccharide was 1.74 and its molecular weight was 56.2 kDa. We provided evidences that JCS1S2 could disrupt angiogenesis both in vitro and in vivo. This sulfated polysaccharide inhibited migration and tube formation of human microvascular endothelial cells (HMEC-1) whereas showed no effect on the cells proliferation. Further study uncovered that JCS1S2 bound to both VEGF (vascular endothelial growth factor) (KD value: 4.82 × 10−9) and VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) (KD value: 1.50 × 10-7) to inactivate VEGFR2 phosphorylation. In addition, JCS1S2 blocked downstream signaling and impaired the expression of VEGF and its transcription factor AP-1 (Activator protein-1). These results demonstrated that JCS1S2 interrupted angiogenesis via blocking VEGF signaling transduction and could be a potential anti-angiogenetic agent for disease treatment.