Retinal neovascularization is a leading cause of human blindness. However, little is known concerning the molecular mechanisms controlling retinal neovascularization in vivo. Here we provide evidence that exposure of a collagen type IV cryptic epitope detected by monoclonal antibody (mAb) HUIV26, delineates sites of vascular bud formation and represents one of the earliest structural remodeling events required before vessel out-growth. Exposure of these cryptic sites was inhibited in matrix metalloproteinase (MMP)-9-deficient but not MMP-2-deficient mice implicating MMP-9 in their exposure. Retinal endothelial cell interactions with the HUIV26 epitopes induced endothelial cell migration, which was blocked by mAb HUIV26. Importantly, subcutaneous administration of mAb HUIV26 potently inhibited retinal angiogenesis in vivo. Taken together, these findings suggest a novel mechanism in which MMP-9 facilitates exposure of HUIV26 cryptic sites, thereby promoting retinal endothelial cell migration and neovascularization in vivo.