Overstimulation of cAMP-activated Cl⁻ secretion can cause secretory diarrhea. Isoliquiritigenin (ISLQ) is a plant-derived chalcone that has a wide range of biological activities. The present study thus aimed to investigate the effect of ISLQ on cAMP-activated Cl⁻ secretion in human intestinal epithelium, especially the underlying mechanism and therapeutic application. Short-circuit current analysis of human intestinal epithelial (T84) cell monolayers revealed that ISLQ dose-dependently inhibited cAMP-activated Cl⁻ secretion with an IC₅₀ of approximately 20 μM. ISLQ had no effect on either basal short-circuit current or Ca²⁺-activated Cl⁻ secretion. Apical Cl⁻ current analysis of T84 cell monolayers indicated that ISLQ blocked mainly the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels, but not other unidentified cAMP-dependent Cl⁻ channels. ISLQ did not affect intracellular cAMP levels or cell viability. ISLQ completely abolished the cholera toxin-induced transepithelial Cl⁻ secretion in T84 cells and reduced the cholera toxin-induced intestinal fluid secretion in mouse closed loop models by 90%. Similarly, ISLQ completely inhibited the cAMP-activated apical Cl⁻ current across monolayers of Madin-Darby Canine Kidney (MDCK) cells and retarded cyst growth in MDCK cyst models by 90%. This study reveals a novel action of ISLQ as a potent CFTR inhibitor with therapeutic potential for treatment of cholera and polycystic kidney disease.