Mucociliary clearance is a primary defence mechanism of the airways consisting of two components, ciliary beating and transepithelial ion transport (I). Specialised chemosensory cholinergic epithelial cells, named brush cells (BC), are involved in regulating various physiological and immunological processes. However, it remains unclear if BC influence I. In murine tracheae, denatonium, a taste receptor agonist, reduced basal I in a concentration-dependent manner (EC 397 µM). The inhibition of bitter taste signalling components with gallein (G subunits), U73122 (phospholipase C), 2-APB (IP3-receptors) or with TPPO (Trpm5, transient receptor potential-melastatin 5 channel) reduced the denatonium effect. Supportively, the I was also diminished in Trpm5 mice. Mecamylamine (nicotinic acetylcholine receptor, nAChR, inhibitor) and amiloride (epithelial sodium channel, ENaC, antagonist) decreased the denatonium effect. Additionally, the inhibition of G subunits (pertussis toxin) reduced the denatonium effect, while an inhibition of phosphodiesterase (IBMX) increased and of adenylate cyclase (forskolin) reversed the denatonium effect. The cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR and the KCNQ1 potassium channel antagonist chromanol 293B both reduced the denatonium effect. Thus, denatonium reduces I via the canonical bitter taste signalling cascade leading to the Trpm5-dependent nAChR-mediated inhibition of ENaC as well as G signalling leading to a reduction in cAMP-dependent I. Therefore, BC activation contributes to the regulation of fluid homeostasis.