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Our previous studies elucidated that hydroxysafflor yellow A (HSYA) exerted anti-inflammatory effects against ischemia stroke by inhibiting TLR4 pathway-mediated signaling transduction. However, only several targets were verified in that limited work. The integrated method of serial affinity chromatography (SAC) and shotgun proteomics analysis (SPA) might be an alternative approach for exploring a potential therapeutic role. SAC was induced to extract specific binding proteins in the brain tissue of 2 h of ischemia stroke mice via HSYA affinity matrices. SPA was conducted by nanoLC-MS/MS, while the identified proteins were mapped on to Gene Ontology and KEGG pathway components analysis. The protection of HSYA for blood-brain barrier in mice with ischemia stroke was assessed with the leakage of Evans Blue. The expression of tight junction proteins of blood-brain barrier: occludin, claudin-5, and ZO-1 were detected with ischemia boundary positive areas staining. The regulation of nonmuscle myosin heavy chain IIA (NMMHC IIA), TLR4-mediated PI3K/AKT/JNK1/2/14-3-3ε/NF-κB p65 signaling pathway were evaluated using western blot analysis. A total of 35 proteins with molecular eights ranging from 27,841.22 to 234,122.79 KD were identified. Gene Ontology annotation and KEGG pathways analysis of the identified proteins were conducted with tight junction and PI3K/AKT signaling pathways. HSYA could significantly reduce the leakage of Evans Blue in mice with ischemia stroke, while attenuating the expression of occludin, claudin-5, and ZO-1. Western blot demonstrated that regulation of NMMHC IIA, TLR4-mediated PI3K/AKT/JNK1/2/14-3-3ε/NF-κB p65 signaling pathway played an essential role in the protective effect of HSYA. The integrated method of SAC and SPA provides the promising explanations for exploring the mechanism underlying blood-brain barrier dysfunction via the tight junction pathway. HSYA could attenuate blood-brain barrier dysfunction in anti-inflammatory patterns in ischemia stroke mice via the tight junction pathway.The integrated method of SAC and SPA was induced to explore specific binding proteins.Bioinformatics annotation showed binding proteins related with tight junction pathway.NMMHC IIA, TLR4/PI3K/Akt/JNK1/2/14-3-3ε pathway participated in BBB dysfunction.HSYA reduced BBB leakage by attenuating occludin, claudin-5, and ZO-1.HSYA attenuated BBB dysfunction in ischemia stroke mice via tight junction pathway.