Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 μg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.