High levels of chenodeoxycholic acid (CDCA) and deoxycholic acid stimulate Cl⁻ secretion in mammalian colonic epithelia. While different second messengers have been implicated in this action, the specific signaling pathway has not been fully delineated. Using human colon carcinoma T84 cells, we elucidated this cascade assessing Cl⁻ transport by measuring I⁻ efflux and short-circuit current (I_sc). CDCA (500 μM) rapidly increases I⁻ efflux, and we confirmed by I_sc that it elicits a larger response when added to the basolateral vs. apical surface. However, preincubation with cytokines increases the monolayer responsiveness to apical addition by 55%. Nystatin permeabilization studies demonstrate that CDCA stimulates an eletrogenic apical Cl⁻ but not a basolateral K⁺ current. Furthermore, CDCA-induced I_sc was inhibited (≥67%) by bumetanide, BaCl₂, and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR_inh-172. CDCA-stimulated I_sc was decreased 43% by the adenylate cyclase inhibitor MDL_12330A and CDCA increases intracellular cAMP concentration. The protein kinase A inhibitor H89 and the microtubule disrupting agent nocodazole, respectively, cause 94 and 47% reductions in CDCA-stimulated I_sc. Immunoprecipitation with CFTR antibodies, followed by sequential immunoblotting with Pan-phospho and CFTR antibodies, shows that CDCA increases CFTR phosphorylation by approximately twofold. The rapidity and side specificity of the response to CDCA imply a membrane-mediated process. While CDCA effects are not blocked by the muscarinic receptor antagonist atropine, T84 cells possess transcript and protein for the bile acid G protein-coupled receptor TGR5. These results demonstrate for the first time that CDCA activates CFTR via a cAMP-PKA pathway involving microtubules and imply that this occurs via a basolateral membrane receptor.