Submucosal glands line the cartilaginous airways and produce most of the antimicrobial mucus that keeps the airways sterile. The glands are defective in cystic fibrosis (CF), but how this impacts airway health remains uncertain. Although most CF mouse strains exhibit mild airway defects, those with the C57Bl/6 genetic background have increased airway pathology and susceptibility to Pseudomonas. Thus, they offer the possibility of studying whether, and if so how, abnormal submucosal gland function contributes to CF airway disease. We used optical methods to study fluid secretion by individual glands in tracheas from normal, wild‐type (WT) mice and from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice (Cftr^m1UNC/Cftr^m1UNC; CF mice). Glands from WT mice qualitatively resembled those in humans by responding to carbachol and vasoactive intestinal peptide (VIP), although the relative rates of VIP‐ and forskolin‐stimulated secretion were much lower in mice than in large mammals. The pharmacology of mouse gland secretion was also similar to that in humans; adding bumetanide or replacement of HCO₃⁻ by Hepes reduced the carbachol response by ∼50%, and this inhibition increased to 80% when both manoeuvres were performed simultaneously. It is important to note that glands from CFTR knockout mice responded to carbachol but did not secrete when exposed to VIP or forskolin, as has been shown previously for glands from CF patients. Tracheal glands from WT and CF mice both had robust secretory responses to electrical field stimulation that were blocked by tetrodotoxin. It is interesting that local irritation of the mucosa using chili pepper oil elicited secretion from WT glands but did not stimulate glands from CF mice. These results clarify the mechanisms of murine submucosal gland secretion and reveal a novel defect in local regulation of glands lacking CFTR which may also compromise airway defence in CF patients.