Increased secretion of colonic epithelial human β defensins (HβDs) has been proposed to contribute to the pathogenesis of ulcerative colitis (UC) through induction of chemokines and the consequent infiltration of mucosal immune cells. Although alterations in the colonic bile acid pool also occur in UC, the roles of bile acids in regulating HβD production are not yet known. Here, we investigated the effects 2 common colonic bile acids on epithelial HβD release; deoxycholic acid (DCA), the most common of the colonic bile acids, and ursodeoxycholic acid (UDCA), which we and others have shown to exert anti-inflammatory actions in in vivo models of disease. HβD release from monolayers of T84 colonic epithelial cells or muscle-stripped sections of human colon mounted in Ussing chambers were measured by ELISA after treatment with DCA (10 – 150 µM) or UDCA (50 – 100 µM). Colonic mβD-1 and mβD-4 mRNA levels from WT and TGR5-/- mice were measured by q-PCR. Use of human and mouse tissue was approved by the Beaumont Hospital Ethics Committee. DCA significantly increased HβD-1 and HβD-2 secretion from T84 cells from 190 ± 28 pg/ml to 413 ± 34 pg/ml and 27 ± 5 pg/ml to 291 ± 2 pg/ml (n = 4; p < 0.01), respectively. Furthermore, UDCA attenuated DCA-stimulated HβD-1 and HBD-2 release to 82 ± 10 pg/ml and 95 ± 17 pg/ml, respectively (n = 4; p < 0.05). Similar effects were seen in ex vivo sections of distal human colon. Similar to DCA, (INT-777, 10 µM) a specific agonist of the bile acid receptor, TGR-5, stimulated both HβDs release from T84 cells and these responses were significantly reduced by UDCA treatment. INT777 (30 μM) failed to increase levels of mβD-1 and 4, orthologues of HβD-1 and 2, respectively, in TGR5-/- but not in WT mice. Finally, a specific inhibitor of NF-κB, BMS-34451 (25 μM) attenuated DCA (150 µM)-induced HβD-2, but not HβD-1, secretion to 27 ± 8 pg/ml (n = 6; p < 0.01). Taken together our data suggest that DCA, likely through activation of TGR5, promotes colonic epithelial HβD secretion. In contrast, UDCA inhibits HβD secretion, an effect which may underlie its anti-inflammatory actions in vivo. Thus, alterations in colonic bile acid pool may influence UC pathogenesis through alterations in HβD production.