Background: Viral and bacterial infections are the most common reasons for asthma exacerbation. Pathogens can be recognized by Toll-like receptors(TLRs) which are importation parts of the innate immunity. However, the role of TLRs in asthma aggravation is unclear. The aim of this study was to investigate the effects of TLRs activation on naïve guinea pigs. Method: Poly(I:C), LPS and Imiquimod, as the agonist of TLR3, TLR4 and TLR7, respectively, were used to mimic specific infections. Animals were treated with PBS or TLRs agonists intranasally for 4 consecutive days. After each exposure the breathing pattern, measured as enhanced pause(Penh), was recorded by whole-body plethysmography. One day after the last challenge, the airway responsiveness to nebulized methacholine was measured using forced oscillation technique. Bronchoalveolar lavage fluid(BALF) was collected to analyze the inflammatory cells. Result: Treatment of LPS and Imiquimod, but not Poly(I:C), induced a significant increase of Penh directly after challenge. Animals exposed to Poly(I:C), LPS or Imiquimod demonstrated significantly increased maximal lung resistance(3.93±0.57; 2.14±0.27 and 4.13±0.60 cm H2O·s·ml-1) compared to PBS(0.76±0.08 cm H2O·s·ml-1). A marked increase of neutrophils was found in the BALF of animals challenged with LPS, whereas no increase of cells was induced by Poly(I:C) and Imiquimod. Conclusion: In this study, TLR4 stimulation increased the neutrophilic inflammation in the airways, TLR4 and TLR7 stimulation caused breathing changes of guinea pigs and whereas activation of TLR3, TLR4 or TLR7 induced AHR. This suggest that the guinea pig is sensitive to different microbial stimulation of the airways.