Otitis media is the most prevalent childhood disease in developed countries. The involvement of Toll-like receptors (TLRs) in otitis media pathophysiology has been implicated by studies in cell lines and association studies of TLR gene polymorphisms. However, precise functions of TLRs in the etiology of otitis media in vivo have not been examined. We investigated the inflammatory response to nontypeable Haemophilus influenzae using a model of otitis media in wild-type, TLR2(- /-) and TLR4(-/ -) mice by gene microarray, qPCR, immunohistochemistry, Western blot analysis and histopathology. Toll-like receptor-2(- /-) and TLR4(- /-) mice exhibited a more profound, persistent inflammation with impaired bacterial clearance compared to controls. While wild-type mice induced tumor necrosis factor-a (TNF) after non-typeable H. influenzae challenge, TLR2(-/-) and TLR4(-/-) mice lack TNF induction in the early phase of otitis media. Moreover, lack of TLR2 resulted in a late increase in IL-10 expression and prolonged failure to clear bacteria. Toll-like receptor-4(-/- ) mice showed impaired early bacterial clearance and loss of TLR2 induction in early otitis media. Our results demonstrate that both TLR2 and TLR4 signalling are critical to the regulation of infection in non-typeable H. influenzae-induced otitis media. Toll-like receptor-4 signalling appears to induce TLR2 expression, and TLR2 activation is critical for bacterial clearance and timely resolution of otitis media.