Background: Excessive mucin secretion in the airway is an important feature of airway inflammatory diseases. MUC5AC expression is regulated by a variety of stimuli such as cytokines. Little is known about the role of interferon (IFN)-γ in MUC5AC expression in human bronchial epithelial cells. Methods: Human pulmonary mucoepidermoid carcinoma cell line (NCI-H292) and normal human bronchial epithelial (NHBE) cells were used to assess the effects of IFN-γ on MUC5AC transcription. Results: Transforming growth factor (TGF)-α and double-stranded RNA (polyI:C)-induced MUC5AC mRNA and protein expression was repressed by IFN-γ in a concentration-dependent manner. IFN-γ showed limited effects on TGF-α and polyI:C-induced activation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). A chromatin immunoprecipitation assay indicated that Sp1 bound to its cognate sequence located on the MUC5AC promoter. The Sp1 inhibitor mithramycin A inhibited MUC5AC mRNA expression, implying a critical role for Sp1 in MUC5AC induction. Importantly, IFN-γ impeded Sp1 binding to the MUC5AC promoter. Conclusions: These results suggest that IFN-γ represses MUC5AC expression, disturbing binding of Sp1 to its target sequences.