Rationale: Mucus overproduction and airway obstruction are common features in airway mucosal inflammation. The mechanism by which LPS induces MUC5AC overexpression, however, has not been fully explored. Objectives: The aims of this study were two-fold; firstly, to examine the ATP-dependent mechanism by which LPS induces MUC5AC gene expression, and secondly, to identify specific molecules which could suppress LPS-induced MUC5AC expression at a G-protein coupled receptor level. Results: Here, we suggest that LPS from Pseudomonas aeruginosa induces MUC5AC overproduction by both an ATP-dependent pathway and an ATP-independent pathway. In addition, we showed that Regulator of G-protein signaling (RGS) 4 plays as a suppressor for ATP-induced MUC5AC expression by interacting with Galphaq in a GTP-dependent manner in vivo. Conclusions: These results give additional insights into the molecular mechanism of negative regulation of mucin overproduction and enhance our understanding of mucus hypersecretion during airway mucosal inflammation.