Mucus obstruction and airway inflammation are key pathological features of chronic airway diseases including cystic fibrosis (CF) and asthma. Previous studies in human primary airway epithelial cultures and transgenic mouse models demonstrated that airway surface dehydration caused by defficient CFTR-mediated Cl+ secretion and/or increased Na+ absorption mediated by epithelial Na+ channels (ENaC) constitutes an important mechanism leading to impaired mucociliary clearance and lung disease in CF.Besides restoration of CFTR function, activation of alternative Cl+ channels may improve airway surface hydration in CF airways. Recent evidence suggests that epithelial Cl+ secretion is increased in Th2-mediated airway inflammation, however, the molecular identity of the underlying Cl+ conductance was not known. SLC26A9 is a member of the SLC26 family of anion transporters predominantly expressed in epithelia of the lung and upper GI tract. Recent studies showed that SLC26A9 functions as a Cl+ channel that contributes to constitutive and cAMP-dependent Cl+ secretion in human bronchial epithelial (HBE) cells. Based on its functional properties in transduced cells and expression pattern in mouse airways, we hypothesized that SLC26A9 may function as an alternative Cl+ channel that may contribute to airway surface liquid (ASL) homeostasis in health and in Th2-mediated airway inflammation.To test this hypothesis, we compared transepithelial ion transport in freshly excised bronchial tissues, lung morphology and airway mucus content in wild-type versus Slc26a9-deficient mice under physiological conditions and after intratracheal instillation of IL-13 to model Th2-mediated airway inflammation. Further, in a human study population of 661 children with asthma (i.e. a prototypical Th2-dominated airway disease) and 658 healthy controls, we tested if polymorphisms in the SLC26A9 gene are associated with asthma.In bioelectric studies in native airway tissues, lack of SLC26A9 had no effect on basal, cAMP-mediated or Ca2+-activated Cl+ conductance (CaCC) under physiological conditions. In airways from wild-type mice, constitutive as well as Ca2+-activated Cl+ secretion were ~2-fold increased after intratracheal instillation of IL-13. The IL-13 induced constitutive Cl+ conductance was completely abrogated, whereas up-regulation of CaCC was preserved in native airway tissues from SLC26A9-deficient mice. While IL-13 induced goblet cell metaplasia and mucus overproduction to similar levels in both genotypes, lack of SLC26A9-mediated Cl+ secretion was associated with airway mucus obstruction in IL-13 treated SLC26A9-deficient mice that did not occur in wild-type controls.Testing of effects of genetic variants within the SLC26A9 locus on asthma risk identified a single nucleotide polymorphisms (SNP) in the 3’UTR of SLC26A9 (rs2282430) that was associated with asthma. In silico analyses predicted that the A allele of rs2282430 strengthens binding of hsa-miR-632 to the SLC26A9 3’UTR. Functional testing of precursor hsa-miR-632 on the wild-type 3’UTR sequence and the SNP in HEK293 cells using luciferase reporter assays demonstrated that hsa-miR-632 reduced luciferase activity in cells co-transfected with the SNP compared with the wild-type 3’UTR. These results suggest that the SNP in the SLC26A9 3’UTR identified in our genetic studies reduced protein expression and indicate that binding of hsa-miR-632 is involved in this process.Our studies demonstrate that SLC26A9 constitutes a Cl+ channel in native airway epithelia that is quiescent under physiological conditions, but constitutively active to promote Cl+ secretion and airway surface hydration in Th2-mediated airway disease. The observation that genetic deletion of SLC26A9 produced significant airway mucus obstruction in allergic airway disease indicates that SLC26A9-mediated Cl+ secretion is essential for maintaining ASL homeostasis required for effective MCC in the presence of mucus hypersecretion. These results suggest that SLC26A9 may serve as a novel therapeutic target to improve airway surface hydration in CF and potentially other mucostatic airway diseases.