The aim of this work was to set up and validate an in vitro model to study a molecular response of an intestinal host cell line (HT29-MTX), to a non-pathogen microflora component. We found that Bacteroides thetaiotaomicron strain VPI-5482 had the capacity to change a specific glycosylation process in HT29-MTX cells via a mechanism that involved a soluble factor. Differentiated HT29-MTX cells were grown in the presence of 20% of spent culture supernatant from the B. thetaiotaomicron during 10 days. Glycosylation processes were followed using a large panel of lectins and analysed using confocal microscopy, western blotting and flow cytometry techniques. Our results show that a B. thetaiotaomicron soluble factor modified specifically the galactosylation pattern of HT29-MTX cells, whereas other glycosylation steps remained mainly unaffected. Further characterization of this soluble factor indicates that it is a heat labile, low molecular weight compound. Reverse transcript-PCR (RT-PCR) analysis was unable to show any significant change in mRNA expression level of the main galactosyltransferases expressed in HT29-MTX cells. By contrast, galactosyltransferase activities dramatically increased in HT29-MTX cells treated by the soluble extract of B. thetaiotaomicron, suggesting a post-translational regulation of these activities. Our in vitro model allowed us to study the cross-talk between a single bacteria and intestinal cells. The galactosylation process appears to be a target of this communication, thus uncovering a new window to study the functional consequences of co-operative symbiotic bacterial-host interactions.