Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel™. We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α 1β 1 and α 2β 1 integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.