To elucidate the regulatory role of protein palmitoylation in blood-brain barrier disruption during cerebral ischemia/reperfusion injury, we performed palmitoylproteomic profiling of cerebral cortical tissues from wild-type and endothelial-specific PEAK1-knockout mice subjected to middle cerebral artery occlusion/reperfusion. Site-specific mapping identified Cys1350 as the primary palmitoylation site of PEAK1 catalyzed by ZDHHC8. Pathway enrichment analysis further revealed that differentially palmitoylated proteins were mainly involved in sphingolipid signaling and ceramide biosynthesis. These findings establish ZDHHC8-mediated PEAK1 palmitoylation as a critical pathogenic regulatory node in ischemic stroke.