Enhanced antibiotic resistance of P. aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus, we developed a co-culture model and report that airway cells increase the resistance of P. aeruginosa to Tobramycin (Tb) by >25-fold compared to P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achieved in the lungs of CF patients. In addition, CF airway cells expressing F508-CFTR significantly enhanced P. aeruginosa biofilm formation, and F508 rescue with WT-CFTR reduced biofilm formation. Iron (Fe) concentration in the bronchial alveolar lavage fluid is elevated in CF, and Fe is known to enhance P. aeruginosa growth. Thus, we investigated whether enhanced biofilm formation on F508-CFTR cells was due to increased Fe release by airway cells. Indeed, airway cells expressing F508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, and Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The F508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.