<p>Astragaloside IV (AS-IV) is the principal active component of <i>Astragalus membranaceus</i> (fisch.) Bge. var. <i>mongholicus</i> (Bge.) Hsiao. This study aims to explore action mechanism of AS-IV for relieving of cerebral ischemic injury in a rat model of middle cerebral artery occlusion reperfusion (MCAO) via proteomics and network pharmacology. Pharmacodynamics experiments showed that AS-IV could effectively alleviate MACO-induced cerebral infarction, preserve the structural integrity of neurons, and promote the formation of Sol bodies. In addition, TMT quantitative proteomics revealed differential proteins (DEPs), e.g., DGKQ, PPT1, Gnai3, Gnal, PLA2G4A, and Ppp2ca. These DEPs might be closely related to AS-IV for the therapeutic effects on ischemic stroke. In combination with network pharmacology, the PLA2G4A was further identified as key target protein of AS-IV ascribed to its involvement in the regulation of inflammatory mediators in the TRP pathway. Ultimately, in <i>vitro</i> validation demonstrated that AS-IV offers neuroprotective effects by targeting the PLA2G4A, reducing the release of arachidonic acid (AA) and COX-2, and facilitating Ca²⁺ inflow into cells. This study provided a scientific basis on development and application of AS-IV for treating ischemic stroke.</p> Graphical abstract <p></p>