Growth factors affect a variety of epithelial functions. We examined the ability of TGF-β to modulate epithelial ion transport and permeability. Filter-grown monolayers of human colonic epithelia, T84 and HT-29 cells, were treated with TGF-β (0.1–100 ng/ml, 15 min–72 h) or infected with an adenoviral vector encoding TGF-β (Ad-TGFβ) for 144 h. Ion transport (i.e., short-circuit current, I sc) and transepithelial resistance (TER) were assessed in Ussing chambers. Neither recombinant TGF-β nor Ad-TGFβ infection affected baseline I sc; however, exposure to ≥1 ng/ml TGF-β led to a significant (30–50%) reduction in the I sc responses to forskolin, vasoactive intestinal peptide, and cholera toxin (agents that evoke Cl− secretion via cAMP mobilization) and to the cell-permeant dibutyryl cAMP. Pharmacological analysis of signaling pathways revealed that the inhibition of cAMP-driven epithelial Cl− secretion by TGF-β was blocked by pretreatment with SB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors of JNK, ERK1/2 MAPK, or phosphatidylinositol 3′-kinase. TGF-β enhanced the barrier function of the treated monolayers by up to threefold as assessed by TER; however, this event was temporally displaced from the altered I sc response, being statistically significant only at 72 h posttreatment. Thus, in addition to TGF-β promotion of epithelial barrier function, we show that this growth factor also reduces responsiveness to cAMP-dependent secretagogues in a chronic manner and speculate that this serves as a braking mechanism to limit secretory enteropathies.