Mucin 5AC (MUC5AC) hypersecretion induces airway narrowing in patients with asthma, which leads to breathing problems. We investigated the regulation of MUC5AC secretion by extracellular matrix (ECM) proteins in human primary airway epithelial cells from patients with asthma. The addition of type IV collagen to three-dimensional cultured human primary airway epithelial cells, which mimics the airway surface, reduced MUC5AC secretion in the medium, while the addition of laminin increased MUC5AC secretion. Furthermore, the addition of fibronectin did not affect MUC5AC secretion. In particular, the repeated addition of a low concentration of type IV collagen demonstrated a cumulative effect on the reduction in MUC5AC secretion. Human primary cells incubated with type IV collagen showed downregulated extracellular signal-regulated kinase (ERK) activity, which induced MUC5AC hypersecretion but did not affect Akt activity. These results suggest that the addition of type IV collagen to the apical surface of primary cells downregulates MUC5AC secretion and has a cumulative effect on MUC5AC secretion which might be effected via the ERK signaling pathway.