Abstract The ocular surface epithelium serves a critical function as the defensive front line of the innate immune system. While the detection of microbes is arguably its most important task, an exaggerated host defense reaction to endogenous bacterial flora may initiate and perpetuate inflammatory mucosal responses. The ability of cells to recognize pathogen-associated molecular patterns (PAMPs) mainly depends on the expression of a family of Toll-like receptors (TLRs). A healthy ocular surface is not inflammatory, even though ocular surface epithelium is in constant contact with bacteria and bacterial products. In this study, we show that human ocular surface epithelial cells, both corneal and conjuctival epithelial cells, respond to viral double-stranded RNA mimic polyI:C to produce pro-inflammatory cytokines through TLR3, while they fail to respond functionally to lipopolysaccharide, a TLR4 ligand. Moreover, human ocular surface epithelium responds to flagellins from ocular pathogenic, but not ocular non-pathogenic bacteria, to produce pro-inflammatory cytokines through TLR5. Thus, ocular surface epithelial cells selectively respond to microbial components and induce limited inflammation; immune-competent cells can recognize microbial components through TLRs and induce the inflammation. The unique innate immune response of the ocular surface epithelium may contribute to its coexistence with commensal bacteria. Inflammatory bowel disease is thought to result from an abnormal response to the gut microbiota. Thus, we also considered the possibility of an association between ocular surface inflammation and a disordered innate immune response. IκBζ is important for TLR signaling, in mice, its knock-out produced severe, spontaneous ocular surface inflammation, the eventual loss of goblet cells, and spontaneous perioral inflammation, suggesting that dysfunction/abnormality of innate immunity can lead to ocular surface inflammation.