Diabetic keratopathy (DK) is an underdiagnosed ocular complication of diabetes mellitus. The changes of ocular immune microenvironment contribute to the pathogenesis of DK, while precise mechanisms remain inadequately understood. Here, we employed single-cell RNA sequencing (scRNA-seq) to elucidate the transcriptional alterations of immune cells from diabetic and healthy control mouse corneas during homeostasis and wound healing. Unbiased clustering analysis unveiled 3 major cell subsets and 11 subdivided cell clusters, including T cells, monocyte lineages, and neutrophil subpopulations. The further sub-clustering analysis demonstrated that T cells exhibited cytotoxicity characteristics in both homeostasis and wound healing of diabetic cornea. Moreover, dendritic cells preferred the migratory and maturation phenotype and may recruit and maintain cytotoxic T cells. Macrophages in diabetic cornea preferred the pro-inflammatory M1 phenotype. Under injury conditions, diabetic corneal neutrophils exhibited a more mature and functional possession of neutrophil extracellular traps (NETs). Furthermore, cell-cell communication revealed that the immune cells exhibited hyperactivation and pro-inflammatory responses, while the monocyte lineages exhibited the activating effect on T cells in diabetic cornea. This study represents the inaugural effort to establish a comprehensive scRNA-Seq transcriptomic profile of corneal immune cells during wound healing in healthy and diabetic mice, which offers a valuable reference for subsequent investigations into the pathological roles of immune cells in DK.