The original hygiene hypothesis suggests that early childhood respiratory infections prior to allergen exposure may decrease the prevalence of allergic diseases. We have recently demonstrated that M. pneumoniae infection prior to allergen exposure reduced allergic responses in mice. However, the molecular mechanisms underlying the protective role of M. pneumoniae in allergic responses particularly airway mucin production remain unclear. Wild-type and TLR2 deficient mice with a respiratory M. pneumoniae infection preceding allergen (ovalbumin) challenge were utilized to determine the regulatory role of TLR2-IFN- signaling pathway in airway mucin expression. Further, air-liquid interface cultures of mouse primary tracheal epithelial cells were performed to examine the effects of IFN- on mucin expression. In wild-type mice, M. pneumoniae prior to allergen challenge significantly reduced airway mucins, but increased IFN-. In sharp contrast, in TLR2 deficient mice, M. pneumoniae prior to allergen challenge resulted in increased mucin protein without a noticeable change of IFN-. In cultured mouse primary tracheal epithelial cells, IFN- was shown to directly inhibit mucin expression in a dose-dependent manner. Our study demonstrates for the first time that a respiratory M. pneumoniae infection prior to allergen challenge reduces airway epithelial mucin expression in part through TLR2-IFN- signaling pathway. A bacterial infection in asthmatic subjects with weakened TLR2-IFN- signaling may result in an exaggerated airway mucin production.