Many vision-threatening diseases are characterized by intraocular neovascularization, (e.g., proliferative diabetic retinopathy and age-related macular degeneration). Although a new therapy with anti-VEGF antibodies is being used to treat these intraocular neovascular disorders, the visual recovery is limited, mainly because of the remnants of fibrovascular tissues. The ideal goal of the treatment is to prevent the invasion of new vessels into the avascular tissue through a matrix barrier. The purpose of this study was to determine the role played by cathepsin L, a matrix degrading enzyme, on intraocular angiogenesis. Used established animal models of retinal and choroidal neovascularization, we demonstrated that an inhibition of cathepsin L by specific inhibitors resulted in a significant decrease of intraocular neovascularization. A similar decrease of neovascularization was found in cathepsin L-deficient mice. Transplantation of bone marrow from cathepsin L-deficient mice into wild-type mice significantly reduced the degree of intraocular neovascularization. In addition, immunocytochemical analyses demonstrated that VE cadherin-positive endothelial progenitor cells, but not CD43-positive or Iba-1-positive cells, were the major cells contributing to the production of cathepsin L. These data indicate that cathepsin L expressed in endothelial progenitor cells plays a critical role in intraocular angiogenesis and suggest a potential therapeutic approach of targeting cathepsin L for neovascular ocular diseases.