Airway mucus hypersecretion is a hallmark of respiratory diseases. Recently, several reports showed that the extracellular microenvironment regulates mucus hypersecretion; however, its mechanism is not well understood. Therefore, we examined the effect of the integrin inactivating peptide FNIII14 on the production and secretion of MUC5AC, a major component of mucus. In this study, NCI-H292 cells, capable of producing mucus, were treated with TGF-α to induce the production of MUC5AC. FNIII14 did not markedly affect the MUC5AC mRNA expression and the phosphorylation of ERK1/2, a major downstream signaling molecule of EGFR, suggesting that FNIII14 does not affect the production system of MUC5AC. However, the amount of intracellular MUC5AC protein was increased by FNIII14 in a concentration-dependent manner. In addition, intracellular MUC5AC increased by anti-integrinβ1 antibody treatment. These results suggested that secretion of airway mucus is regulated by extracellular matrix and integrins. Furthermore, fluorescence immunostaining revealed that MUC5AC did not co-localize with endoplasmic reticulum or Golgi markers, suggesting that FNIII14 may inhibit the exocytosis system. These findings may lead to the proposal of a new pharmacological mechanism for regulating airway mucus secretion by integrin inactivation.