Probiotics are living microorganisms which, when ingested in adequate amounts, exert health benefits toward the host. For instance, probiotics might act through reinforcement of the intestinal epithelial barrier function. The goal of the present study was to determine whether Lactobacillus casei DN-114 001 could abrogate the increase in paracellular permeability induced by enteropathogenic Escherichia coli. We used the human colon T84 cell line infected with a wild-type enteropathogenic E. coli (strain E2348/69). Paracellular permeability was followed by monitoring transepithelial electrical resistance variations and by observing zonula occludens-1 distribution. Two infection procedures were used: co-incubation (the pathogenic and probiotic strains were simultaneously incubated with T84 cells) and post-infection (the probiotic was added in the presence of pathogenic bacteria 3 h after the beginning of the infection). We also investigated the effect of L. casei on enteropathogenic E. coli adhesion. L. casei DN-114 001 inhibited, in a dose-dependent-manner, the decrease in enteropathogenic E. coli-induced transepithelial electrical resistance and zonula occludens-1 redistribution using two different infection procedures. However, L. casei did not inhibit pathogenic strain adhesion. L. casei DN-114 001 inhibited the increase in EPEC-induced paracellular permeability. This property could partially explain the previously observed health benefits of this probiotic for human natural defenses, such as those associated with prevention of diarrhea.