Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFTR_inh-172, was used to create a CF model with its own control to test if loss of CFTR-Cl⁻ conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-Cl⁻ conductance for 3–5 days resulted in significant increase in IL-8 secretion at basal (P = 0.006) and in response to 10⁹ Pseudomonas (P = 0.0001), a fourfold decrease in Smad3 expression (P = 0.02), a threefold increase in RhoA expression, and increased NF-κB nuclear translocation upon TNF-α/IL-1β stimulation (P < 0.000001). CFTR inhibition by CFTR_inh-172 over this period does not increase epithelial sodium channel activity, so lack of Cl⁻ conductance alone can mimic the inflammatory CF phenotype. CFTR_inh-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTEo⁻ pCEP-R and 16HBE14o⁻ AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFTR_inh-172 effects on cytokines are not direct. Five-day treatment with CFTR_inh-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung.