Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl− efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ influx via the epithelial Na+ channel (ENaC). In cells expressing wt-CFTR, ambroxol increased the Cl- conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl− transport was measured by Cl− efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl− efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α-ENaC, and of the CFTR protein. No significant difference was observed in β-ENaC after exposure to ambroxol, whereas mRNA expression of γ-ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca2+ concentration. Upregulation of CFTR and enhanced Cl− efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients.