Corneal neovascularization (CNV) is a common sight-threatening pathology that can be induced by a variety of inflammatory and angiogenic stimuli. Current CNV treatments include anti-inflammatory drugs and antibody-based inhibitors of vascular endothelial growth factor (VEGF). However, these are not always effective and novel therapeutic approaches are needed. Previous work has indicated a role for nucleolin (NCL) in VEGF-mediated neoangiogenesis in a suture-induced CNV model. The major goal for this current study is to test the effect of AS1411, a NCL-binding DNA aptamer that has reached human clinical trials, on neovascularization in a murine model of VEGF-mediated CNV. Our results show that topical administration of AS1411 can significantly inhibit corneal neovascularization in this model. Mechanistic studies indicate that AS1411 reduces the VEGF-stimulated proliferation, migration, and tube formation of primary cells obtained from human limbus stroma (HLSC). AS1411 treatment also significantly reduced VEGF-stimulated induction of miR-21 and miR-221 in HLSC, suggesting a role for these pro-angiogenic miRNAs in mediating the effects of AS1411 in this system. In sum, this new research further supports a role for NCL in the molecular etiology of CNV and identifies AS1411 as a potential anti-angiogenic CNV treatment that works by a novel mechanism of action.