Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Since glucose is a nutrient present in the airways of patients with bacterial airway infections and since insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by qPCR, immunoblot, immunofluorescence, and glucose uptake assays. PI3K/Akt signaling and CFTR activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed the Glut4 transporter and that application of insulin stimulated cytochalasin B inhibitable glucose uptake consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared to that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.