Cyclic AMP-activated intestinal Cl⁻ secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl⁻ secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl⁻ secretion in human intestinal epithelial (T84) cells with IC₅₀ of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl⁻ current showed that diclofenac reversibly inhibited CFTR Cl⁻ channel activity (IC₅₀∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na⁺-K⁺ ATPases and Na⁺-K⁺-Cl⁻ cotransporters, but inhibited cAMP-activated basolateral K⁺ channels with IC₅₀ of ∼3 µM. In addition, diclofenac suppressed Ca²⁺-activated Cl⁻ channels, inwardly rectifying Cl⁻ channels, and Ca²⁺-activated basolateral K⁺ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl⁻ secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca²⁺-activated Cl⁻ secretion by inhibiting both apical Cl⁻ channels and basolateral K⁺ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl⁻.