Corneal neovascularization is a significant cause of vision loss, often resulting in corneal clouding and chronic inflammation. Shark cartilage is widely recognized as a significant natural source of anti-angiogenic compounds. Our previous studies have shown that a polypeptide from white-spotted catshark ( Bonnet) has the potential to inhibit the angiogenesis of breast tumors. This study applied this peptide (SAIF) to a corneal alkali injury model to assess its effect on corneal neovascularization. Results revealed that SAIF inhibits endothelial cell proliferation, migration, and tube formation. SAIF inhibited VEGF-induced angiogenesis in the matrigel plug. Using the corneal alkali injury model, SAIF significantly inhibited corneal vascular neovascularization in mice. We found that SAIF not only significantly inhibited the upregulation of pro-angiogenic factors such as VEGF, bFGF, and PDGF expression induced by alkali injury, but also promoted the expression of anti-angiogenesis factor PEDF. Moreover, we also analyzed the MMPs and TIMPs involved in extracellular matrix (ECM) remodeling, angiogenesis, and lymphangiogenesis. We found that SAIF treatment inhibited the expression of pro-angiogenic factors like MMP1, MMP2, MMP3, MMP9, MMP13, and MMP14, and promoted the expression of anti-angiogenesis factors such as MMP7, TIMP1, TIMP2, and TIMP3. In conclusion, SAIF acts as an anti-angiogenic factor to inhibit the proliferation, migration, and tube formation of endothelial cells, inhibit pro-angiogenic factors, promote anti-angiogenic factors, and regulate the expression of MMPs, ultimately inhibiting corneal neovascularization.