Mucus secretion is often uncontrolled in many airway inflammatory diseases of humans. Identifying the regulatory pathway(s) of mucus gene expression, mucus overproduction, and hypersecretion is important to alleviate airway inflammation in these diseases. However, the regulatory signaling pathway controlling mucus overproduction has not been fully identified yet. In this study, we report that the ATP/P2Y2 complex secretes many cytokines and chemokines to regulate airway inflammation, among which IL-1 receptor antagonist (IL-1ra) downregulates MUC5AC gene expression via the inhibition of Gαq-induced Ca2+ signaling. IL-1ra inhibited IL-1α protein expression and secretion, and vice versa. Interestingly, ATP/P2Y2-induced IL-1ra and IL-1α secretion were both mediated by PLCβ3. A dominant-negative mutation in the PDZ-binding domain of PLCβ3 inhibited ATP/P2Y2-induced IL-1ra and IL-1α secretion. IL-1α in the presence of the ATP/P2Y2 complex activated the ERK1/2 pathway in a greater degree and for a longer duration than the ATP/P2Y2 complex itself, which was dramatically inhibited by IL-1ra. These findings suggest that secreted IL-1ra exhibits a regulatory effect on ATP/P2Y2-induced MUC5AC gene expression, through inhibition of IL-1α secretion, to maintain the mucus homeostasis in the airway. Therefore, IL-1ra could be an excellent modality for regulating inflamed airway microenvironments in respiratory diseases.