Background/Aims: Macrolide antibiotics are effective drugs in chronic bronchiolitis and chronic rhinosinusitis with mucus hypersecretion. However, the mechanism of action is unclear. This study was designed to investigate the effect of azithromycin (AZM; 15-membered) and midecamycin acetate (MDM; 16-membered) on MUC5AC and MUC2 gene expression and secretion from human airway epithelial cells. The effects of the two macrolides on tumor necrosis factor-α (TNF-α) release were also examined. Methods: Confluent NCI-H292 human mucoepidermoid airways epithelial cells were pretreated with AZM or MDM for 2 h and then stimulated with 200 nmol/l phorbol 12-myristate 13-acetate (PMA) for 8 h. The MUC5AC and MUC2 gene expression was measured by real-time quantitative RT-PCR. Total mucin in culture supernatants was measured using enzyme-linked lectin assay. Enzyme-linked immunosorbent assay was used to determine MUC5AC, MUC2 and TNF-α released by the cells. Results: AZM and MDM attenuated PMA-induced MUC5AC and MUC2 gene and protein expression in NCI-H292 cells. They also suppressed PMA-mediated TNF-α in the cells. Conclusion: The present study demonstrates that AZM and MDM suppress the synthesis of mucin and TNF-α from human airway epithelial cells.