MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as major regulators of the protein content of a cell. In the most part, miRNAs negatively regulate target mRNA expression, with sets of miRNAs predicted to regulate certain signaling pathways. We have demonstrated that the non-coding RNA expression profiles of endobronchial brushings is altered in people with cystic fibrosis (CF) compared to those without CF (1, 2). How this impacts on CF has important implications for our growing understanding of the pathophysiology of CF lung disease and the development of new therapeutics to treat its pulmonary manifestations (3-5). Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the F508del CFTR mutation. Here the role of miRNAs that are up regulated in vivo in bronchial brushings from individuals carrying one or two F508del CFTR alleles will be discussed (6). In particular studies that investigate miRNA-mediated regulation of CFTR expression and function will be presented including in vitro validation studies in the CFBE41o- cell line and assessment of the impact of defective chloride ion conductance, genotype and Pseudomonas colonization status on miRNA expression. The results from overexpression and inhibition studies performed using premiRs or antimiRs, respectively, and a CFTR 3’UTR luciferase reporter gene will be shown. Collectively the data show that miR-145, miR-223 and miR-494 are up regulated in CF versus non-CF bronchial brushings and cell lines; in F508del CFTR homozygotoes vs. heterozygotes; in subjects positive for P. aeruginosa and; in cells treated with a CFTR inhibitor or IL-1β. Reciprocal down or up regulation of CFTR gene and/or protein expression was observed following miRNA manipulation and direct miRNA/target relationships demonstrated via a reporter system containing a wild type or mutated full length CFTR 3’UTR. The conclusions from this work are that increased expression of miR-145, miR-223 and miR-494 in vivo in bronchial epithelium of individuals carrying the Fdel508 CFTR mutation correlates with decreased CFTR expression and that defective CFTR function, Pseudomonas colonization and inflammation may affect miRNA expression and contribute to the regulation of Fdel508 CFTR..