Purpose Anti-vascular endothelial growth factor-A (VEGF-A) therapy has shown promise for treating newly-formed corneal vessels, but has little effect on established vessels. VEGF-B is a potent survival factor for vascular endothelial cells. VEGF-B deficiency reduced survival of blood vessels in a murine model of corneal neovascularization (CoNV). We developed an anti-VEGF-B antibody fragment (scFv format) and tested its activity on growing and established vessels in a rat model of CoNV. Methods CoNV was induced in male and female SD rats (12 week - 1 year old) by superficial cautery with silver nitrate. Topical anti-VEGF-B scFv therapy (5 x 5 µg eye drops per day for 14 days) was commenced one day after cautery to determine the effect on growing vessels, or 14 days after cautery to determine the effect on established vessels. In a subset of animals, an additional subconjunctival injection (SCINJ) of 50 µg scFv on days 1 and 8 of treatment was applied. Vessels were perfused with haematoxylin and the cornea was flatmounted. The percentage of the cornea covered by vessels was determined with NIH ImageJ. Data were analysed by Kruskal-Wallis test and Mann-Whitney U with correction for multiple testing. Results After topical therapy the corneal neovascular area was not significantly different between untreated, control scFv treated and anti-VEGF-B scFv treated animals (growing vessels p=0.46, established vessels p=0.86). Topical anti-VEGF-B scFv therapy supplemented with SCINJ in the established vessel group significantly reduced the area of corneal vessels (12.9%, SD±3.6, n=21) when compared with untreated (20.5%, SD±7, n=19) or control scFv treated (22.5%, SD±3.2, n=9) animals (p<0.001). Conclusions Anti-VEGF-B scFv therapy caused regression of established vessels in a rat model of CoNV. Combining anti-VEGF-B treatment with anti-VEGF-A therapy could be useful in the treatment of human CoNV.