Disruption of the intestinal microbiota by reducing the number of bacterial species of Lactobacillus sp. and Bifidobacterium sp., simultaneous colonization by pathogenic strains of Escherichia coli and excessive inflammatory process leads to disruption of the integrity of the intestinal barrier of the colon. Under such conditions, the expression of the protein zonula occludens (ZO-1), one of the key proteins of intercellular solid compounds (protein binding complexes that regulate the selective movement of solutes through the epithelium), decreases, resulting in a disruption of the integrity of the intestinal barrier, and is associated with the development of inflammatory bowel disease. Consequently, there is an increase in sulfide metabolism, which further contributes to the deterioration of the immunopathological process. The aim of this study was to examine the influence of probiotic bacteria (Lactobacillus reuteri) on the modulation of the epithelial barrier structure and colonization of the enteropathogenic strain of Escherichia coli on the inflammatory-conditioned colorectal adenocarcinoma cell line, Caco-2, which was grown under hypoxic conditions and treated with sodium sulfide medium and proinflammatory cytokines (IFN-γ, TNF-α and IL-1β). Fluorescently labeled probiotic bacteria (L. reuteri) and enteropathogenic bacteria E. coli 3014 were implanted into Caco-2 cells at different incubation times. Fluorescently labeled monoclonal antibodies for the ZO-1 protein were then added to the cultures. Analysis of ZO-1 protein expression indicated an increasing trend in the presence of sodium sulfide, a decreasing trend in the presence of the enteropathogenic bacterium E. coli, and an increasing trend in the presence of L. reuteri. The results suggest that the enteropathogenic bacterium E. coli 3014 negatively affects the integrity of the epithelial barrier while L. reuteri, as a probiotic bacterium, affects the reconstitution of the inflammatory-damaged epithelial structure.