Proliferative retinopathies are associated with abnormal angiogenesis that can result in visual impairment or vision loss. The tight junction complex regulates blood-retinal barrier integrity; however, its role in proliferative retinopathies is still at an early stage. Here, we employed human retinal endothelial cells (HRMVECs), and a mouse model of oxygen-induced retinopathy (OIR) to investigate the impact of IL-33 signaling on tight junction disintegration and pathological angiogenesis. Our experimental findings demonstrate that IL-33 induces ZO-1 serine/threonine phosphorylation and tight junction disruption in HRMVECs. In addition, mass-spectroscopy (MS) analysis revealed that treating of HRMVECs with IL-33 induces ZO-1 phosphorylation at Thr861 residue. Furthermore, we observed that NOX1-PKC- signaling modulates IL-33-induced ZO-1 phosphorylation and tight junction integrity in HRMVECs. We also observed that IL-33 depletion significantly reduces OIR-induced NOX1-PKC-ZO-1 signaling, vascular leakage, and pathological retinal neovascularization in the ischemic retina. We also observed that the NOX1-specific inhibitor, fluoflavine (ML-090), attenuated OIR-induced NADPH oxidase activity and pathological retinal neovascularization in the ischemic retina. Thus, we infer that IL-33-mediated NOX1-PKC-ZO-1 signaling regulates ischemia-induced retinal endothelial cell tight junction disruption and retinal neovascularization.