The intestinal barrier function depends on an adequate response to pathogens by the epithelium. Toll-like receptor 3 (TLR-3) recognizes double-stranded RNA, a virus-associated molecular pattern. Activation of TLR-3 with Poly(I:C), a synthetic agonist, modulates tissue repair and permeability in other epithelia; however, the effects of local luminal TLR-3 agonists on gut barrier function are unknown. The aim of this investigation was to evaluate short-term effects of Poly(I:C) on rat ileal and colonic permeability ex vivo. We also studied the acute effects of intrarectal administration of Poly(I:C) on colonic barrier function. Ileum tissues displayed decreased transepithelial electrical resistance (TEER) 1 h after incubation with 200 μg/mL Poly(I:C); however, the mucosa-to-serosa transit of macromolecules (4.4 and 40 kDa dextrans - TD4.4 and FD40, respectively) remained unchanged. Conversely, colon tissue preparations stimulated with 200 μg/mL Poly(I:C) showed a decreased thinning of the mucosal layer after 2 h and a decreased transit of FD40 after 3 h, in comparison to controls. There was no change in colonic TEER after 3 h of treatment. In addition, colon tissue taken from rats 6 h after an intrarectal administration of 100 μg Poly(I:C) also showed decreased permeability to FD40 in the everted gut sac assay at 3 h post-extraction. Tissue morphology remained unchanged. Our results suggest that an acute exposure to Poly(I:C) reduces colon permeability to macromolecules but increases ileum permeability to electrolytes/small molecules ex vivo. Although the mechanism associated to these effects needs further investigation, to our knowledge this is the first report of a direct effect of a TLR-3 ligand in intestinal barrier function and may be of significance to understand region-specific interactions between gut mucosa and microbiota.