Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler’s diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins.(R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC 50 down to∼ 5 nM.(R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC 50 down to 0.1 mg/kg.(R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed> 90% oral bioavailability with sustained therapeutic serum levels for> 4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas,(R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.—Cil, O., Phuan, P.-W., Gillespie, AM, Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, NC, Lin, R., Donowitz, M., Verkman, AS Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.