Purpose It was previously demonstrated that opticin (OPTC) inhibits the collagen-induced promotion of bioactivities of human retinal vascular endothelial cells (hRVECs). The present in vivo study aimed to further investigate the regulatory role of opticin in vitreous collagen-mediated retinal neovascularization and to elucidate its regulatory mechanisms with regard to integrin α2-I domain–GXXGER complex formation and RhoA/ROCK1 signal change. The regulatory role of Mg2+ on integrin α2-I domain–GXXGER complex formation in the above process was also investigated. Methods The zebrafish model of hypoxia-induced retinopathy was established, and OPTC-overexpressing plasmids were intravitreally injected to assess the antiangiogenesis effect of opticin. The regulatory role of opticin in integrin α2-I domain–GXXGER complex formation in vivo was analyzed by mass spectrometry. The mRNA and protein expression of RhoA/ROCK1 were examined. The concentration of Mg2+ as an activator of the integrin α2-I domain–GXXGER complex was measured. Solid-phase binding assays were performed to investigate the interference of opticin in integrin α2 collagen binding and the regulatory role of Mg2+ in that process. Results Opticin and OPTC-overexpressing plasmid injection reduced retinal neovascularization in the zebrafish model of hypoxia-induced retinopathy. Mass spectrometry revealed that opticin could inhibit integrin α2-I domain–GXXGER complex formation. The Mg2+ concentration was also decreased by opticin, which was another indication of the complex activation. Injection of OPTC-overexpressing plasmids inhibited mRNA and the protein expression of RhoA/ROCK1 in the zebrafish model of hypoxia-induced retinopathy. The solid-phase binding assay revealed that opticin could block integrin α2–collagen I binding in the presence of Mg2+. Conclusions Opticin exerts its antiangiogenesis effect by interfering in the Mg2+-modulated integrin α2-I domain–collagen complex formation and suppressing the downstream RhoA/ ROCK1 signaling pathway.