Bile acids are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases, probably resulting from the aspiration of gastroesophageal refluxate. The effects of bile acids on airway epithelium ion transport function have not yet been investigated. The effects of the taurine- conjugated secondary bile acid, taurodeoxycholic acid, TDCA (25 μM), on basal ion transport was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). Data given as mean ± S.E.M. Statistics were generated using the Student’s paired t-test or one-way ANOVA analysis followed by Tukey’s post-hoc test which compares all pairs of groups. P values of ≤ 0.05 were considered to be statistically significant. Our results show that acute (5 min) basolateral TDCA treatment of Calu-3 cells stimulated basal Isc by 39 ± 7% (p = 0.0001, n = 13). The Isc responses to TDCA were abolished in Cl- -free Krebs solution (n = 4). Furthermore, TDCA produced a 32 ± 9% (p = 0.043, n = 5) increase in CFTR Isc that was abolished by pre-treatment with CFTRinh172. We also found that TDCA increases Cl- secretion through calcium-activated chloride (CaCC) channels by 18 ± 7% (p = 0.01, n = 4). In addition, when Na+/K+ ATPase generated currents were measured in Ussing chambers, acute treatment with TDCA increased Na+/K+ pump activity by 13 ± 3% (p = 0.0005, n = 9), while pre-treatment with ouabain eliminated this effect. Acute treatment with TDCA resulted in a rapid 88 ± 12% (p = 0.0002, n = 8) increase in intracellular calcium mobilization, which was abolished in Ca2+ -free buffer indicating that TDCA induced a cellular influx of Ca2+. Furthermore, TDCA increased cAMP levels by 42 ± 14% in Calu-3 cells (p = 0.04, n = 8). Preliminary data from TGR5 knockdown studies suggest that TGR5 is the membrane receptor involved in TDCA induced Cl- secretion in Calu-3 cells. In summary, we show for the first time that low concentrations of bile acids can modulate Cl- secretion in airway epithelial cells and this effect is dependent upon both the duration and sidedness of exposure to the bile acid.