Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA, lithocholic acid (LCA), stimulate Cl^- secretion in human colonic T84 cells (AJP 305:C447-56, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50µM, 15') drastically (>90%) inhibited forskolin-stimulated short-circuit current (I_sc), it did not alter carbachol -stimulated Isc. LCA did not alter basal Isc, transepithelial resistance, cell viability or cytotoxicity. LCA's inhibitory effect was dose-dependent, acted faster from the apical membrane, rapid and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues, 8Br-cAMP, lubiprostone or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA-specific, since CDCA, cholic acid or taurodeoxycholic acid did not alter forskolin or carbachol action. While LCA alone had no effect on [cAMP]_i, it decreased forskolin-stimulated [cAMP]_i by 90%. Although LCA caused a small increase in [Ca²⁺]_i, chelation by BAPTA-AM did not reverse LCA's effect on I_sc. LCA action does not appear to involve known BA receptors, FXR, VDR, M3 or TGR5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD98059. Surprisingly PD98059 did not reverse LCA's effect on Isc. Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical CFTR Cl^- current, and inhibits a basolateral K⁺ current. In summary, 50µM LCA greatly inhibits cAMP-stimulated Cl^- secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.