Increasing body of evidence shows that danger-associated molecular patterns (DAMPs) play a proinflammatory role in the pathogenesis of airway obstructive diseases, such as severe asthma and chronic obstructive pulmonary disease. NLRP3 inflammasome is a molecular platform that activates the caspase-1 pathway in response to danger signals. ATP and S100 proteins are newly identified DAMPs that are accumulated in inflammatory airways. We have previously demonstrated that S1008, S100A9, and S100A12 induce production and secretion of MUC5AC, a major mucin in conducting airway mucosa. The purpose of this study was to determine the involvement of NLRP3 inflammasome in and the contribution of ATP to the S100 protein-induced MUC5AC production by NCI-H292 mucoepidermoid carcinoma cells. Stimulation with either ATP or S100A12 alone led to MUC5AC production, accompanied by increases in NLRP3 synthesis, activation of NF-B and its reporter plasmid, active caspase-1, and proIL-1 at comparable levels. Simultaneous treatment with both stimuli led to increased MUC5AC production in an additive manner, with more markedly increase in co-localization of NLRP3 and caspase-1 than either treatment, suggesting an overlapping signal transduction pathway from both stimuli. NLRP3 siRNA and all pharmacological inhibitors for NLRP3, NF-B, and caspase-1 nearly completely inhibited the ATP- or S100A12-mediated MUC5AC production and/or NF-B reporter activity. Thus, these two inflammatory mediators independently contribute to MUC5AC production in airway epithelial cells commonly through the NLRP3 inflammsome activation.