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. Fourteen distinct clusters were identified, including myeloid cells (neutrophils, monocytes, macrophages), dendritic cells (DC), and lymphoid cells (B, T, γδT, ILC2, and NK) lineages. Novel neutrophil [lipocalin (Lcn2) high and low), and MHCIIlo macrophage (MP) clusters were identified. More than 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 macrophage), Apoe (monocyte), Cd209a (macrophage), Cst3 (cDC1), and IL4i1 in migratory DC (mDC). ILC2 expresses 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 mDC (CCR7, FASCIN1) located on or within the conjunctival epithelium. Monocyte, macrophage, cDC1 and IL-13/IL-5+ ILC2 were located below the conjunctival epithelium and goblet cells. This study found distinct immune cell populations in the conjunctiva and identified cells expressing genes with known homeostatic and immunoregulatory functions.