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Ischemic stroke, the most common type, has threatened human life and health. The treatment options for ischemic stroke are limited due to the complexity of the pathological process and cellular information. Therefore, acute ischemic stroke rats were established by middle cerebral artery occlusion (MCAO), and the cell populations in the cortex of MCAO rats were identified utilizing single-cell RNA sequencing (scRNA-seq). We identified 21 brain clusters with cell-type specific gene expression patterns and cell subpopulations, as well as 42 marker genes representing different cell subpopulations. The number of cells in clusters 0–3 increased significantly in the MCAO group compared to the sham group, and nine cell subpopulations exhibited remarkable differences in the number of genes. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the top 40 differentially expressed genes (DEGs) in the six cell subpopulations with significant differences. The results indicated that the biological processes and signaling pathways are involved in different cell subpopulations. In conclusion, scRNA-seq revealed the diversity of cell differentiation and the unique information of cell subpopulations in the cortex of rats with acute ischemic stroke, providing a novel insight for exploring the pathological process and drug discovery in the stroke.