Tenascin-C is expressed in choroidal neovascular (CNV) membranes in eyes with age-related macular degeneration (AMD). However, its role in the pathogenesis of CNV remains to be elucidated. Here we investigated the role of tenascin-C in CNV formation. In immunofluorescence analyses, tenascin-C co-stained with α-SMA, pan-cytokeratin, CD31, CD34, and integrin αV in the CNV membranes of patients with AMD and a mouse model of laser-induced CNV. A marked increase in the expression of tenascin-C mRNA and protein was observed 3 days after laser photocoagulation in the mouse CNV model. Tenascin-C was also shown to promote proliferation and inhibit adhesion of human retinal pigment epithelial (hRPE) cells in vitro. Moreover, tenascin-C promoted proliferation, adhesion, migration, and tube formation in human microvascular endothelial cells (HMVECs); these functions were, however, blocked by cilengitide, an integrin αV inhibitor. Exposure to TGF-β2 increased tenascin-C expression in hRPE cells. Conditioned media harvested from TGF-β2-treated hRPE cell cultures enhanced HMVEC proliferation and tube formation, which were inhibited by pretreatment with tenascin-C siRNA. The CNV volume was significantly reduced in tenascin-C knockout mice and tenascin-C siRNA-injected mice. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of CNV via integrin αV in a paracrine manner. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of CNV development associated with AMD.