Our previous studies have shown that prior exposure of respiratory epithelial cells to an aqueous-trapped solution of DE (DE_as) enhances the susceptibility to Influenza infections. Here we examined the effect of DE_as on the toll-like receptor 3 (TLR3) pathway, which is responsible for the recognition of and response to viruses and double-stranded RNA. Flow cytometric and confocal microscopy analysis showed that TLR3 is predominantly expressed in the cytoplasm of respiratory epithelial cells. To examine the effect of DE on TLR3 expression and function, differentiated human bronchial or nasal epithelial cells as well as A549 cells were exposed to DE_as and then infected with Influenza A or treated with polyriboinosinic acid:polyribocytidylic acid (poly(I:C)), a synthetic form of dsRNA. Exposure to DE_as prior to infection with Influenza or stimulation with poly(I:C) significantly up-regulated the expression of TLR3. Additionally, pre-exposure to DE_as significantly increased the poly(I:C)-induced expression of IL-6. Overexpression of a dominant negative mutant form of TRAF6 (dnTRAF6) reversed the effects of DE_as on poly(I:C)-induced IL-6 expression, suggesting that the response was TLR3-dependent. Similarly, pre-exposure to DE_as significantly increased nuclear levels of IRF3 and the expression of IFN- in response to poly(I:C). Pre-treatment with wortmannin, a specific inhibitor of PI3 kinase, was able to abate the effect of DE_as on poly(I:C)-induced IFN- expression. Taken together these results indicate that exposure of respiratory epithelial cells to DE_as could potentially alter the response to viral infections by increasing the expression and function of TLR3.