Airway serous secretion is essential for the maintenance of mucociliary transport in airway mucosa, which is responsible for the upregulation of mucosal immunity. Although there are many articles concerning the importance of Toll-like receptors (TLRs) in airway immune systems, the direct relationship between TLRs and airway serous secretion has not been well investigated. Here, we focused on whether TLR5 ligand flagellin, which is one of the components of Pseudomonas aeruginosa, is involved in the upregulation of airway serous secretion. Freshly isolated swine tracheal submucosal gland cells were prepared and the standard patch-clamp technique was applied for measurements of the whole cell ionic responses of these cells. Flagellin showed potentiating effects on these oscillatory currents induced by physiologically relevant low doses of ACh in a dose-dependent manner. These potentiating effects were TLR5-dependent but TLR4-independent. Both nitric oxide (NO) synthase inhibitors and cGMP-dependent protein kinase (cGK) inhibitors abolished these flagellin-induced potentiating effects. Further, TLR5 was abundantly expressed on tracheal submucosal glands. Flagellin/TLR5 signaling further accelerated the intracellular NO synthesis induced by ACh. These findings suggest that TLR5 takes part in the airway mucosal defense systems as a unique endogenous potentiator of airway serous secretions and that NO/cGMP/cGK signaling is involved in this rapid potentiation by TLR5 signaling.