Epithelial cells are often first responders to microbial invasion, resulting in release of inflammatory mediators such as cytokines and chemokines and subsequent recruitment of immune cells. Several strains from the genus Lactobacillus have been attributed probiotic properties and some of them have been shown to be able to modulate the immune response in vitro and in vivo. L. rhamnosus GR-1, a known probiotic strain was allowed to co-culture with an uroepithelial cell line challenged with heat-killed pathogenic E. coli, resulting in a synergistic up-regulation of the transcription factor NF-κB and increased release of the pro-inflammatory cytokine TNF. Although this L. rhamnosus strain was a poor inducer of uroepithelial NF-κB alone compared to heat-killed E. coli, together with this pathogenic stimulus, it efficiently elicited an epithelial immune response. This effect was greatly reduced if using non-viable lactobacilli suggesting secretion of the active substance. Secreted proteins were isolated and partially mimicked the effect found with viable lactobacilli. Potentiation of the host immune response towards pathogenic E. coli at an early stage using lactobacilli products could facilitate the removal of undesired microbes by activation of the NFκB transcription factor and consequently, epithelial immunity.