Objective: The objective of this study was to provide insight into the molecular mechanisms of acute ischemic stroke (IS) through gene expression profiling analysis. Methods: The gene chip data of ischemic stroke patients and the control group in the GEO database were downloaded. Differential genes were analyzed by GEO2R software and the R language. Metascape was used to analyze gene ontology (GO) and gene sets of enzyme and pathway analysis (Reactome). The hub genes were identified using the STRING online analysis tool and Cytoscape software. Finally, quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed to verify the expression of DEGs in the peripheral blood of IS patients. Results: A 20-gene profile was identified in the whole blood of ischemic stroke patients from the gene expression datasets. Gene annotation and signal pathway analysis showed that these differentially expressed genes were mainly involved in the response of neutrophils to extraneous biological stimulation, the response of cytokines to organic anion production, transport to fatty acids, and the migration of neutrophils, etc. Five genes (mmp-9, OLFM4, ORM1, FOLR3, and ARG1) were identified as hub gene by Cytoscape and MCODE software. Finally, ARG1 and MMP9 mRNA overexpressed in peripheral blood of IS patients compared with normal control confirmed by RT-PCR. Conclusion: The dysregulation of several genes may involve the process of ischemic stroke (IS). Our finding provided clues for exploring mechanisms and developing novel diagnostic and therapeutic strategies for IS.