Airway submucosal glands have been proposed as a primary site for initiating and sustaining airway disease in cystic fibrosis (CF). However, it has been difficult to define the role of CFTR in gland fluid secretion because of concerns in interpreting experiments on diseased CF human airways subjected to chronic infection and inflammation. Here, we test the role of CFTR in gland fluid secretion by using a selective CFTR inhibitor (CFTR_inh‐172) in pig and human airways. Measurements of single‐gland fluid secretion rates showed inhibition of both cholinergic and cAMP‐stimulated fluid secretion by CFTR_inh‐172. Secreted fluid [Na⁺] and [Cl^–] measured by fluorescence ratio imaging were 101 and 116 mM, respectively, and not significantly altered by secretory agonists or CFTR inhibition. Gland fluid pH was 7.1 and reduced by 0.4 units after CFTR inhibition. Gland fluid viscosity, determined by photobleaching of FITC‐dextran, was threefold increased in pilocarpine‐stimulated gland fluid after CFTR inhibition, and protein concentration was increased from 12 to 20 mg/ml. Our data provide strong evidence that gland fluid secretion is CFTR‐dependent. The relatively hyper‐viscous and acidic fluid secretions produced by acute CFTR inhibition support a role for defective gland function in CF lung disease and provide a rational basis for pharmacological creation of a large animal model of CF.