Bile acids (BAs) play a complex role in regulating colonic fluid secretion. We showed that chenodeoxycholic acid (CDCA), but not lithocholic acid (LCA), stimulates Cl- secretion via cAMP-PKA activation of CFTR in human colonic T84 cells (AJP 305:C447-56, 2013). Further, LCA inhibits forskolin-, CDCA- or 8-Br-cAMP-induced increases in short-circuit current, Isc, and forskolin-induced [cAMP]i (Gastro. 146:5:S785, 2014). Here, we further delineate the processes underlying the contrasting actions of CDCA (500µM) and LCA (50µM) in T84 cells. LCA’s inhibition is rapid, specific to cAMP secretagogues, and slowly reversible (2h: 50% recovery), while that of CDCA is rapidly reversible (min). In nystatin-permeabilized cells, LCA stimulates an apical CFTR Cl- current and inhibits a basolateral K+ current. LCA (1-24h) has no effects on transepithelial resistance (TER), dilution potentials (cation selectivity), permeability (measured by dextran flux), or occludin localization. In contrast, CDCA (1-24h) decreases TER, decreases cation selectivity, increases permeability, induces IL-8 release, and redistributes occludin. Proinflammatory cytokines (PiC: TNFa+IFNy+IL1ß) also increase permeability, TER and IL-8; PiC and CDCA effects are additive. LCA reverses CDCA or PiC-induced changes in permeability and IL-8 release but not those of TER. BA actions did not involve the BA receptors, FXR or TGR5, or muscarinic M3 receptors. The EGFR inhibitor, AG1478, did not reverse LCA’s attenuation of Cl- secretion. However, both AG1478 and EGFR siRNA attenuate (>67%) CDCA’s action. Interestingly, while both LCA and CDCA increase ERK1/2 phosphorylation >2-fold, that was blocked by the MEK inhibitor PD98059, the latter did not inhibit CDCA or LCA-dependent ΔIsc. Although LCA and CDCA increase [Ca2+]i, only CDCA action was blocked by the chelator BAPTA. CDCA increases [IP3] and requires IP3 receptor activation. Since CDCA action involves PKA and [Ca2+]i , further exploration revealed that inhibition of the exchange protein activated by cAMP (EPAC) with ESI09 (10µM) reduced CDCA’s secretory response (ΔIsc, µA/cm2; CDCA: 17±2; CDCA+ESI09: 5±1; n=6; p<0.05). Inhibition of both PKA (H89, 30µM) and EPAC blocks CDCA response by 97% (n=5). H89 and ESI09 decrease CDCA-induced EGFR phosphorylation (n=2), implying that cAMP mediates CDCA’s transactivation of EGFR. Downstream effector of EPAC, Ras-related protein 2, but not Rap1A and Rho kinase, may be involved in CDCA action. In summary, CDCA and LCA have opposite effects in T84 cells: CDCA stimulates Cl- secretion via complex signaling (PKA+EPAC+Ca2++EGFR), causes barrier dysfunction and releases IL-8, while LCA attenuates cAMP-stimulated Cl- secretion, restores barrier integrity and suppresses IL-8 release. The interplay of these two BAs in regulating colonic function in health and inflammatory diseases could provide useful insights to novel therapeutic strategies.