Previous investigation showed that polyphenols abundantly found in many plants could inhibit Cl(-) secretion. The present study was aimed to investigate the effect of phenol containing xanthone derivatives on cAMP-activated intestinal Cl(-) secretion and evaluate potential benefits of these compounds in the treatment of cholera. Four hydroxy xanthones were synthesized via oxidative coupling reaction of the corresponding ortho-hydroxybenzoic acids and hydroxyphenols. Short-circuit current and apical Cl(-) current measurements across monolayers of human intestinal epithelial (T84) cell and Fisher rat thyroid cells transfected with human CFTR (FRT-hCFTR cell) were performed to determine the effect of hydroxyxanthones on cAMP-activated Cl(-) secretion. Intracellular cAMP was measured by immunoassay methods. Anti-diarrheal efficacy was evaluated using closed loop model of cholera. Among the tested xanthones, 1,3,6-trihydroxyxanthone (THX-001) was found to be the most potent derivative in the inhibition of cAMP-activated Cl(-) secretion across T84 cell monolayers (IC(50)~100M). Electrophysiological analysis of T84 cells and FRT-hCFTR cells revealed that THX-001 targeted two distinct cAMP-activated Cl(-) channels in the apical membrane of T84 cells, namely, CFTR and inward rectifying Cl(-) channel (IRC). In contrast, THX-001 had no effect on intracellular cAMP levels in these cells. Importantly, THX-001 completely abolished cholera toxin-induced Cl(-) secretion across T84 cell monolayers and significantly inhibited cholera toxin-induced intestinal fluid secretion in mouse closed loop models. This study revealed that hydroxyxanthone represents another chemical class of polyphenolic compounds that may hold promise as anti-secretory therapy for cholera.