Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the pro-inflammatory cytokine IL1β via activation of CREB in the NCI-H292 cancer cell line and NFB in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis-site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NFB subunits to the IL1β-induced upregulation of MUC5AC. Data show that ligand binding of IL1β to the IL1β receptor is required to increase MUC5AC mRNA abundance. ChIP analyses show direct binding of CREB to the previously identified CRE site and binding of p65 and p50 subunits to a novel NFB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NFB site in the distal promoter. p50 binds to both NFB sites at 1 h following IL1β exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus, IL1β activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that Dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL1β-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.