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Zonula occludens-1 (ZO-1) is a tight junction protein in the cerebrovascular endothelium, responsible for blood–brain barrier function. Hydroxysafflor yellow A (HSYA) is a major ingredient of safflower (<i>Carthamus tinctorius</i> L.) with antioxidative activity. This study investigated whether HSYA protected ZO-1 by targeting ROS-generating NADPH oxidases (NOXs). HSYA administration reduced cerebral vascular leakage with ZO-1 protection in mice after photothrombotic stroke, largely due to suppression of ROS-associated inflammation. In LPS-stimulated brain microvascular endothelial cells, HSYA increased the ratio of NAD⁺/NADH to restore Sirt1 induction, which bound to Von Hippel–Lindau to promote HIF-1<i>α</i>degradation. NOX2 was the predominant isoform of NOXs in endothelial cells and HIF-1<i>α</i> transcriptionally upregulated p47phox and Nox2 subunits for the assembly of the NOX2 complex, but the signaling cascades were blocked by HSYA via HIF-1<i>α</i> inactivation. When oxidate stress impaired ZO-1 protein, HSYA attenuated carbonyl modification and prevented ZO-1 protein from 20S proteasomal degradation, eventually protecting endothelial integrity. In microvascular ZO-1 deficient mice, we further confirmed that HSYA protected cerebrovascular integrity and attenuated ischemic injury in a manner that was dependent on ZO-1 protection. HSYA blocked HIF-1<i>α</i>/NOX2 signaling cascades to protect ZO-1 stability, suggestive of a potential therapeutic strategy against ischemic brain injury.