Abstract Body: Introduction: Ischemic stroke (IS) significantly alters brain cellular composition and gene expression, particularly in glial cells like astrocytes (ASTs), oligodendrocytes (OLs), and oligodendrocyte precursor cells (OPCs). While these cells' roles in neural repair are recognized, the precise molecular mechanisms remain unclear. This study aims to elucidate IS-induced transcriptomic changes in ASTs, OLs, and OPCs using single-cell RNA sequencing (scRNA-seq) of human brain tissues. We hypothesize that IS triggers distinct transcriptomic alterations in glial cells, crucial for post-stroke neural repair, which can be mapped to specific subclusters with unique functions. Methods: We performed scRNA-seq on five human brain tissue samples: three controls from craniocerebral trauma patients and two from IS patients. Tissues were collected from the frontotemporal regions within 48 hours of stroke onset. We analyzed 23,638 cells, identifying eight cell types. Differential gene expression, subcluster analysis, and trajectory analysis were focused on ASTs, OLs, and OPCs to explore the functional implications of the observed changes. Results: IS resulted in a marked increase in ASTs and OPCs, while OLs decreased in number. ASTs displayed significant transcriptional alterations, particularly in subclusters associated with synaptic signaling and axon development. Key upregulated genes included NMNAT2 (neuroprotection), PPM1H (involved in cellular stress response), and NRP1, (axonal guidance and the formation of synapses). OLs showed reduced presence post-IS, with subclusters enriched for synaptic regulation pathways. OPCs exhibited significant proliferation post-IS, with subclusters displaying divergent developmental trajectories, suggesting altered differentiation pathways due to stroke.Pseudotime analysis revealed distinct gene expression dynamics, with early-stage OPCs (Cluster 1) showing greater stemness compared to later-stage cells (Cluster 2). Conclusion: This study provides a detailed transcriptomic atlas of glial cell changes after IS, highlighting the critical roles of astrocytes, OLs, and OPCs in post-stroke brain repair. IS alters both cellular composition and gene expression, particularly in pathways related to synaptic transmission and neuronal development. These findings deepen our understanding of post-stroke recovery and suggest potential therapeutic targets for neural repair.