Purpose - To investigate the expression and/or function of toll-like receptors (TLRs) and antimicrobial peptides (AMPs) in dry eye inflammation. Methods - Experimental dry eye (EDE) was induced in C57BL/6 mice and TLR mRNA and protein expression were determined at the ocular surface and lacrimal gland. TLR agonist cocktail was applied to the ocular surface in untreated (UT), corneal scratched, and EDE mice. The corneal expression of cathelin-related antimicrobial peptide (CRAMP; human LL-37 orthologue), and mouse beta defensin (mBD)-3 and -4 (human BD-2 orthologue) was compared. LL-37, hBD-2, TLR4, 5, and TLR9 mRNA expression was examined in patients with dysfunctional tear syndrome (DTS) via conjunctival impression cytology. Murine central corneal thickness (CCT) and inflammatory cell recruitment into the stroma was determined by in vivo imaging. Results - EDE upregulated TLR2-4 and 9 mRNA expression in the palpebral conjunctiva and with the exception of TLR4, a similar expression, occurred in the corneal epithelium. TLR2 and 5 were upregulated in lacrimal gland and overall, there was a corresponding change in TLR protein. EDE decreased CRAMP mRNA and protein. hBD-2 and TLR9 expression were modulated in DTS subjects. Topical TLR agonist increased inflammatory cells recruitment and CCT in mice with a cornea scratch. In EDE, TLR agonist treatment downregulated corneal mBD-4 protein caused corneal epithelial loss, and stromal ulceration resulting in decreased CCT. Conclusions - DTS modulates the expression of TLR and CRAMP and topical application of TLR agonists in EDE mice resulted in corneal epithelial loss and thinning. These results suggest that TLRs are involved in DTS inflammation.