We aimed to determine if toll-like receptor (TLR) expression is modulated in response to dry eye-associated conditions and in dry eye syndrome (DES). Primary human corneal epithelial cells (HCEC), an SV40 HCEC cell line or a normal human conjunctival epithelial cell line (IOBA-NHC) were cultured under hyperosmolar stress (HOS) (400–500 mOsm/kg) or with DES associated cytokines (IL-1α/β, TNFα or TGFβ) at concentrations ranging from 1 to 1000 ng/ml for up to 24 h. Epithelial cells were harvested from a human cornea organ culture model following 24 h of desiccation. Conjunctival impression cytology samples were harvested from subjects with DES and age and gender-matched normal subjects. TLR4, TLR5 or TLR9 mRNA or protein was examined by quantitative RT-PCR, western blotting or flow cytometry. TLR functionality was evaluated in terms of addition of TLR agonists and quantitation of secreted inflammatory cytokines by the use of ELISA and Luminex assays. In SV40 HCEC, HOS significantly increased TLR4 by 8.18 fold, decreased TLR9 by 0.58 fold, but had no effect on TLR5 mRNA expression. TLR4 and TLR9 protein were decreased by 67.7% and 72% respectively. TLR4 mRNA was also significantly up-regulated by up to 9.70 and 3.36 fold in primary HCEC and IOBA-NHC respectively. DES associated cytokines had no effect on TLR4, TLR5 and TLR9 expression. In response to desiccation, TLR4 and TLR5 mRNA were significantly up-regulated by 4.81 and 2.51 fold respectively, while TLR9 mRNA was down-regulated by 0.86 fold in HCEC. A similar trend for TLR4 and TLR9 protein was observed. TLR9 mRNA was significantly down-regulated by almost 59.5% in DES subjects. In conclusion, changes in TLR expression occur in dry eye and could have an important role in ocular surface susceptibility to inflammation and infection.