Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by altered bowel habits and recurrent abdominal pain. Throughout the GI tract, patients with diarrhea-predominant IBS (IBS-D) exhibit increased permeability linked to the downregulation and redistribution of several tight junction (TJ)-related proteins. Stress exacerbates IBS symptoms and increases intestinal permeability in both human and rodents. Corticotropin-releasing factor (CRF) via its CRF1 receptor is a key player in the stress-related alterations of gut function. We previously established that peripheral injection of cortagine, a selective CRF1 agonist, recapitulates the cardinal symptoms of IBS-D in rodents. Recent evidence also suggests that lubiprostone, an agonist of the ClC-2 chloride channel approved to treat IBS with constipation, promotes repair of barrier properties. Our aim was to assess whether an acute therapeutic treatment with lubiprostone could prevent the cortagine-induced alterations of murine ileal epithelial barrier. Female Balb/c mice (8-9 weeks, n=3-9) were injected intraperitoneally with cortagine (30 μg/kg) in sterile water (0.2 ml) and 30 min later, lubiprostone (3, 10 or 30 μg/kg) or vehicle (MCT, medium-chain triglyceride) was given per os (PO, 100 μl). Mice were euthanized 1h, 2h and 4h after MCT and 2hr after lubiprostone. Segments of ileum were collected for qPCR assays and in vitro permeability studies in Ussing chambers. After seromuscular stripping, mounting of tissue in the chambers and stabilization for 45 min, the short-circuit current (Isc), transepithelial electrical resistance (TER) and mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) were recorded as indices of intestinal epithelial barrier function. Cortagine increased FD4 ileal flux at 1h and 2h post-injection (0.20 ± 0.03 and 0.14 ± 0.05 vs 0.08 ± 0.01 nmol/min/cm2 for vehicle, p<0.001 and p<0.05, respectively, one-way ANOVA, Dunnett’s post hoc), but did not affect the TER or Isc. Lubiprostone at 30 μg/kg prevented the increase in ileal epithelial permeability induced by cortagine (0.08 ± 0.04 vs 0.14 ± 0.05 nmol/min/cm2, p<0.05, unpaired t test) while lower doses had no effect. Lubiprostone increased the ileal Isc similarly at all doses, but did not affect the TER in cortagine-treated mice. In MCT-treated mice, cortagine did not affect the gene expression of TJ proteins, but exhibited a trend to increase IL1β and IL6 gene expression. Lubiprostone restored the basal levels of IL1β and IL6 and up-regulated the gene expression of occludin and JAM-A. These results support a protective role of lubiprostone (30 μg/kg PO) against increased ileal epithelial permeability in a mouse model of IBS-D like symptoms, and may represent a potential new therapeutic venue for IBS-D patients.