Immune cells in the exposed conjunctiva mucosa defend against environmental and microbial stresses. Expression profiling by single cell RNA sequencing was performed to identify conjunctival immune cell populations expressing homeostatic and regulatory genes. Sixteen distinct clusters were identified, including myeloid cell (neutrophils, monocytes, macrophages), dendritic cell (DC) and lymphoid cell (B, T, gdT, ILC2 and NK) lineages. Novel neutrophil [lipocalin (Lcn) high and low), MHCIIlo macrophage (MP), and Retnla DC2 clusters were identified. Approximately half of the cells map to myeloid and dendritic cell populations with differential expression profiles that include genes with homeostatic and regulatory functions: SerpinB2 (MHCIIlo MP), Apoe (monocytes), Cd209a (MP), Cst3 (cDC1) and IL4i1/IL4r1 in migratory DC (mDC). ILC2 express the goblet cell trophic factor IL-13. Suppressed inflammatory and activated anti-inflammatory/regulatory pathways were observed in certain myeloid and DC populations. Confocal immunolocalization of identity markers showed cDC2 (Retnla), cDC1 (Cst1) and mDC (Cc7, Fscn1, Il4ra) located on or within the conjunctival epithelium. Monocyte, macrophage and IL-13/IL-5+ ILC2 were located below the conjunctival epithelium and goblet cells. This study revealed more specific cell identity markers and distinct immune cell populations expressing genes with known homeostatic and immunoregulatory functions.