Intestinal epithelial cell differentiation is a complex process in which many different signaling pathways are likely involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to inhibit enterocyte differentiation; however, the mechanisms through which cAMP/PKA signaling modulates differentiation of human intestinal epithelial cells are still not well understood. Herein, we report that: (1) treatment of Caco-2/15 cells with 8Br-cAMP repressed sucrase–isomaltase and villin protein expression and strongly attenuated morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush border formation; (2) treatment of confluent Caco-2/15 cells with 8Br-cAMP led to a strong decrease in F-actin localized at cell–cell contact sites along with a reduced amount of E-cadherin and catenins, but not of ZO-1, at cell–cell interfaces concomitant with a decreased association of these proteins with the actin cytoskeleton; (3) inhibition of PKA by H89 prevented disruption of adherens junctions by extracellular calcium depletion; (4) treatment of Caco-2/15 cells with 8Br-cAMP prevented the recruitment and activation of p85/PI-3K to E-cadherin-mediated cell–cell contacts, an important event in the assembly of adherens junctions and differentiation of these cells; (5) E-cadherin appears to be phosphorylated on serine in vivo in a PKA-dependent mechanism. Conclusion: Our studies show that cAMP/PKA signaling negatively regulates adherens junction integrity as well as morphological and functional differentiation of intestinal epithelial cells. © 2005 Wiley-Liss, Inc.