Glucocorticoids strongly influence the mucosal-defense functions performed by the bronchial epithelium and inhaled corticosteroids (ICS) are critical in the treatment of patients with inflammatory airway diseases such as asthma, COPD and cystic fibrosis. A common pathology associated with these diseases is reduced mucociliary clearance, a defense mechanism involving the coordinated transport of salt, water and mucus by the bronchial epithelium, ultimately leading to the retention of pathogens and particles in the airways and to further disease progression. In the present study, we investigated the role of hydrocortisone (HC) in differentiation and development of the ion transport phenotype of normal human bronchial epithelial (NHBE) cells under air-liquid interface (ALI) conditions. NHBE cells differentiated in the absence of HC (HC0) showed significantly less benzamil-sensitive short-circuit current compared to controls as well as a reduced response after stimulation with the selective β₂-adrenergic receptor (AR) agonist salbutamol. Apical membrane localization of ENaCα subunits were similarly reduced in HC0 cells compared to controls, supporting a role of HC in the trafficking and density of sodium channels in the plasma membrane. Additionally, glucocorticoid exposure during differentiation regulated the transcription of CFTR and β₂-AR mRNAs and appeared to be necessary for the expression of CFTR-dependent anion secretion in response to β₂-agonists. HC had no significant effect on surface cell differentiation but did modulate the expression of mucin mRNAs. These findings indicated that glucocorticoids support mucosal defense by regulating critical transport pathways essential for effective mucociliary clearance.