Bile acids, produced by liver and classically known for their roles in facilitating digestion, are also known to have important roles in regulating several aspects of intestinal physiology, such as epithelial barrier and transport function and mucosal immune responses Although, levels of bile acids can also be elevated in the airways in several diseases, such as gastro-oesophageal reflux disease and cystic fibrosis, little is known about their effects on lung physiology and pathophysiology. The aim of this study was to investigate the effects of bile acids on cytokine secretion from human airway epithelial cells. Calu-3 lung epithelial cells were cultured on permeable supports and transepithelial electrical resistance (Rte) was recorded as a measure of barrier integrity. Cytokine secretion into the bathing medium was measured by sandwich ELISA and stimulated with Poly (I:C) and TNF-α, specific agonists for TLR3 and TNFR1, respectively. Lactate dehydrogenase (LDH) release was used as a measure of cell viability. Statistical analysis was performed by One-way ANOVA with the Tukey-Kramer post-test. p values < 0.05 were considered statistically significant. The lipophillic bile acid, DCA (200 µM), increased secretion of the proinflammatory cytokines IL-8, IL-6 and TNF-α to 220.1 + 23.5% (n = 10, p < 0.05), 572.6 + 185.5 % (n = 9, p < 0.05), and 431.6 + 223.2 % (n=4), compared to unstimulated Calu-3 cells. In contrast, the more hydrophillic bile acid, UDCA (200 µM), was without effect, as were the taurine-conjugated derivatives of DCA and UDCA. Furthermore, DCA (200 µM) significantly enhanced IL-8 and IL-6 release from Calu-3 cells in response to both the TLR3 and TNFR1 agonists, Poly (I:C) (25 µg/ml) and TNF-α (100 ng/ml), respectively. In contrast, UDCA (500 µM) enhanced TNF-α-induced IL-8 release but did not alter either IL-6 or IL-8 release in response to Poly (I:C). Intriguingly, we found that at lower concentrations, both DCA and UDCA specifically inhibited TLR3-induced TNF-α release. Poly (I:C)-stimulated TNF-α release from Calu-3 cells was reduced from 13.4 + 2.1 pg/ml in control cells to 4.5 + 1.0 pg/ml in DCA (50 µM)-treated cells (n = 7, p < 0.05), whereas UDCA (1 µM) also attenuated Poly (I:C)-stimulated TNF-α release to 5.4 + 1.6 pg/ml (n = 7, p < 0.05). Taken together, these data indicate that bile acids play a complex role in regulating cytokine secretion from Calu-3 cells. Whether they exert anti-inflammatory or proinflammatory actions depends on the hydrophobicity of the particular bile acid in question and the concentration present. These findings are important to developing our understanding of the role that bile acids may play in the pathogenesis of lung inflammation.