We aimed to determine the anti-angiogenic effect of tenomodulin (TeM) on retinal neovascularization in an oxygen-induced retinopathy (OIR) mouse model. OIR was induced in C57BL/6 mice by exposing seven-day-old mice to 75% oxygen for five days followed by room air for five days. Control mice were exposed to room air from birth until postnatal day 17. Mice received intravitreal injections of 1 mu g of TeM in one eye and PBS in the contralateral eye at P7 before being exposed to 75% oxygen. Eyes were collected at postnatal day 17. Retinal blood vessel patterns were visualized by fluorescein angiography. We quantified the number of neovascular nuclei that were present beyond the inner limiting membrane (ILM) using histological methods with a masked approach. Furthermore, double immunohistochemical staining of TeM was performed on retinas to identify nuclei protruding into the vitreous cavity. Western blot was used to detect exogenous TeM protein. The central nonperfusion area (NPA, mm(2)) of TeM-injected eyes was significantly different from that of OIR and PBS-injected eyes, and the number of nuclei in new blood vessels breaking through the ILM in each retinal cross-section significantly differed from that of OIR eyes and PBS-injected control eyes. Cellular nuclei of new blood vessels protruding into the vitreous cavity were also observed in TeM-injected retinas by immunohistochemistry. Western blotting revealed a 16-kDa immunoreactive protein, indicating incorporation of an exogenous TeM fragment into the retina. Our data shows that TeM can effectively inhibit pathological angiogenesis in mouse eyes; indicating its potential role in prevention and treatment of ocular neovascularization.