In this study, we analyzed the mechanism of hydroxysafflor yellow A (HSYA) for treating ischemic stroke (IS) based on network pharmacology tools, and verified the kernel targets via animal experiments. The targets of HSYA were collected via PharmMapper server and the IS-related targets were searched using Genecards, Online Mendelian Inheritance in Man, Therapeutic Target, and Disgenet databases. The targets identified from the above two steps were overlapped to acquire candidate targets involved in the effects of HSYA for treating IS. Subsequently, the Database for Annotation, Visualization, and Integrated Discovery was used for gene ontology analysis and the Kyoto encyclopedia of genes and genomes pathway analysis. Cytoscape 3.7.1 was applied to establish the component-target-pathway network. Potential core targets were obtained by protein-protein interaction analysis. Furthermore, Autodock Vina was used to identify core genes, and animal experiments was used to verify the expression level of core genes. On the basis of the modified neurologic severity score and the results of 2,3,5-Triphenyltetrazolium chloride and Hematoxylin-eosin staining, we confirmed that HSYA reduced the infarct volume in rats and protected neuronal cells in the hippocampal region after IS. Western blot and immunohistochemical staining showed that HSYA increased the expression of epidermal growth factor receptor, hypoxia inducible factor 1 alpha, and endothelial nitric oxide synthase (P < 0.05). The effects of HSYA on IS are mediated through several targets and pathways related to the regulation of oxidative stress and the renewal of cell and blood vessels while improving post-ischemic brain impairment.Copyright © 2021 Elsevier B.V. All rights reserved.