INTRODUCTION. Bicarbonate secretion in the airways contributes to maintaining acid-base homeostasis and the pulmonary function. Disruption of bicarbonate entry into the airway surface liquid (ASL), as has been observed in cystic fibrosis, leads to several defects in lung function at least in part, due to accumulation of a thick and sticky mucus. At a molecular level, apical CFTR channels transport Cl- and HCO3- from the intracellular compartment to the lumen. CFTR also favors bicarbonate secretion by exchange through apical pendrin (SLC26A4), while the contribution of the calcium-activated chloride channel TMEM16A to HCO3- secretion is still unclear. On the other hand, the molecular mechanism involved in HCO3- uptake from the basolateral compartment has been poorly studied. Here, we investigated the expression levels and role of potential basolateral bicarbonate transporters in human and murine airway epithelial cells. RESULTS. RT-PCR revealed that different members of the SLC4A family, including SLC4A4/NBCe1, were expressed in murine and human airway epithelial cells and immunostaining showed the basolateral location of SLC4A4. In primary human airway epithelial cells (hAECs), inhibition of SLC4A4 using Na+-free solution or the pharmacological inhibitor S0859 (30 µM) prevented basolateral HCO3- uptake, measured by intracellular pH (pHi) and short-circuit currents (Isc) in Ussing chambers. SLC4A4 inhibition also decreased both resting and forskolin-stimulated ASL pH. When used ex vivo on mice tracheas, S0859 induced a decrease in Isc only in the presence of HCO3- and Slc4a4 was necessary for Ca2+-stimulated HCO3- secretion. Finally, Slc4a4-/- mice showed a reduced survival, decreased weight, defects in tracheal cartilage formation and bared signs of muco-obstructive airway disease with damaged epithelium and mucus accumulation. Additionally, tracheas from Slc4a4-/- animals showed a decreased mucociliary clearance ability when compared to tracheas from wild type animals. CONCLUSION. Using human primary airway epithelial cells and mouse models, our study revealed a central role for the HCO3- transporter SLC4A4 in airway homeostasis. Importantly, it also showed a previously unreported lung phenotype in mice lacking the HCO3- transporter Slc4a4-/-, characterized by mucus accumulation and reduced mucociliary clearance, providing a new animal model and potential therapeutic targets for obstructive airway diseases.