Abstract Body (Do not enter title and authors here): Introduction: Ischemic stroke (IS) is a pressing global health concern. Diabetes mellitus not only heightens the risk of IS but also aggravates its outcomes. There is established evidence that neutrophil infiltration is involved in secondary inflammation after stroke. In the present study, we aimed to examine the factors that contribute to the deterioration of IS in diabetic mice focusing on mechanisms underlying immune cell infiltration by single cell RNA sequencing (scRNAseq) of brain before and after stroke. Methods: Experimental stroke was induced using the distal middle cerebral artery occlusion (dMCAO) model. db/db mice were used to model type 2 diabetes and obesity, while db/+ mice served as euglycemic controls. Mononuclear cells from ipsilateral brains were isolated 3 days post-dMCAO or sham surgery with the Percoll gradient following enzymatic digestion. Transcriptomic analysis was performed using scRNAseq from db/+ sham, db/db sham, db/+ stroke, and db/db stroke. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) staining were conducted to validate the changes detected in the transcriptome results. Results: Prior to the onset of stroke, db/db mice exhibited transcriptomic changes in endothelial cells and myeloid cells, including macrophages and microglia, mirroring patterns of changes after stroke. Subcluster analysis of macrophages revealed that a subset, enriched for genes associated with tissue damage and repair mechanisms (Lgals3, Arg1, Fabp5, Thbs1), constituted approximately half of the macrophage population in both of the diabetic non-stroke group and the non-diabetic stroke group. Enrichment analysis indicated a strong association of this macrophage subset with neutrophil degranulation. The results of temporal cell type changes shown in FACS revealed a heightened neutrophil presence in the brain parenchyma of diabetic mice before stroke, and further elevated after stroke compared to control mice. Specifically, the increased expression of anaphylatoxin receptor C3aR and complement C3 shown by IF corroborated with the transcriptomic signature, supporting a role of complement activation, which is closely associated with neutrophil migration. Conclusions: Our findings provide valuable insights into the mechanisms whereby diabetes potentially escalates neutrophil infiltration post-stroke, emphasizing the need for therapeutic interventions targeting myeloid cells to reduce neuroinflammation among diabetic stroke patients.