Aims: Anti-vascular endothelial growth factor agents effectively treat age-related macular degeneration and myopic choroidal neovascularization (CNV). Tissue plasminogen activator (tPA), a fibrinolytic compound, is used as an adjuvant to displace submacular hemorrhage and to treat type 2 CNV. The purpose of this study was to investigate in in vitro and in vivo experiments the antiangiogenic impact of tPA itself. Methods: The impact of tPA on the proliferation of human umbilical vein endothelial cells (HUVECs) was assessed by an XTT assay [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide]. A basic fibroblast growth factor-impregnated gelatin hydrogel sheet was implanted into the rabbit cornea to induce corneal neovascularization. Immediately postoperatively, tPA or buffered saline solution (control) was injected intravitreally. Results: The growth and viability of the HUVECs were unaffected by tPA at clinical concentrations. In the control group, the mean lengths of the new vessels were 1.0 ± 0.41, 1.6 ± 0.75, and 3.6 ± 2.1 mm at weeks 1, 2, and 4, respectively. In contrast, tPA significantly (p < 0.01) reduced the corneal neovascularization. Conclusion: Although tPA has no direct impact on the vascular endothelial cells in vitro, the fibrinolytic effects of tPA might markedly suppress neovascularization in vivo.