PURPOSE. We tested the ability of an antibody fragment with specificity for vascular endothelial growth factor-B (VEGF-B) to regress nascent and established corneal blood vessels in the rat. METHODS. A single chain variable antibody fragment (scFv) with specificity for VEGF-B was engineered from the 2H10 hybridoma. Binding to rat, mouse, and human VEGF-B was confirmed by surface plasmon resonance. Activity of the anti–VEGF-B scFv on developing and established corneal blood vessels was assessed following unilateral superficial cautery in male and female outbred Sprague Dawley rats. Groups (untreated, control scFv-treated, or anti– VEGF-B scFv-treated) comprised 6 to 22 rats. Treatment consisted of 5 μL scFv, 1 mg/mL, applied topically five times per day for 14 days, or two subconjunctival injections, 50 μg scFv each, applied 7 days apart, or combined topical and subconjunctival treatment. Corneal vessel area was quantified on hematoxylin-stained corneal flat-mounts, and groups were compared using the Mann-Whitney U test, with post hoc Bonferroni correction. Immunohistochemistry for cleaved caspase-3 was performed. RESULTS. Topical anti–VEGF-B scFv therapy alone did not regress corneal blood vessels significantly (P > 0.05). Subconjunctival injection and combined treatment regressed 14-day established corneal blood vessels (25% reduction in vessel area [P = 0.04] and 37% reduction in vessel area [P < 0.001], respectively, compared to results in untreated controls). Cleaved caspase-3 was identified in vascular endothelial cells of anti–VEGF-B scFv-treated corneas. In scFv-treated rats, corneal endothelial cell function was maintained to 12 weeks after treatment and a normal blink reflex was present. CONCLUSIONS. The anti–VEGF-B scFv significantly regressed established but not developing corneal blood vessels in rats.