Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., -Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33) Tg+ mice. As compared with IL-33/Tg+ mice, IL-33/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major cytokine. As compared with IL-33/Tg+ mice, IL-33/Tg+ mice had significantly reduced levels of -associated gene signatures (, and ), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33/Tg+ mice, although the IL-33/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33/Tg+ mice compared with IL-33/Tg+ mice. Taken together, our data indicate that although IL-33 modulates inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.Copyright © 2020 by The American Association of Immunologists, Inc.