Background: Tight-junction (TJ) proteins regulate paracellular permeability. Gut permeability can be modulated by commensal microbiota. Manipulation of the gut microbiota with antibiotics like bacitracin and neomycin turned out to be useful for the treatment of diarrhoea induced by Clostridium difficile or chemotherapy drugs.Aim: To evaluate the effects of the microbiota depletion evoked by the oral administration of neomycin and bacitracin on the intestinal permeability and expression of TJ proteins in mice.Methods: Mice received neomycin and bacitracin orally for 7 days. Intestinal permeability was measured by the fluoresceinisothiocyanate-dextran (FITC-dextran) method. The gene expression of TJ proteins in the intestine was determined by real time-PCR.Results: FITC-dextran levels in serum were reduced by half in antibiotic-treated mice, indicating a reduction of intestinal permeability. Antibiotics increased the expression of zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A, and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon.Conclusion: The combination of neomycin and bacitracin reduce intestinal permeability and increase the gene expression of ZO-1, junctional adhesion molecule A (JAM-A), and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon.