Aim: The changes produced on the bacterial surface of Bifidobacteria cells when they are grown in bile were compared with those provoked by bile added to bacteria grown in the absence of bile. Methods and Results: The adhesive properties, the zeta potential and the lipid composition of Bifidobacterial strains, isolated from human faeces and grown in MRS medium, were determined. Bacteria grown in MRS with bile showed a loss of adherence and autoaggregation in correlation with a decrease in the surface hydrophobicity in comparison to those grown in MRS without bile, concomitant with the absence of two glycolipids, the increase of sugar content and minor changes in fatty acid composition. The surface changes caused by bile shock on bacteria grown in bile-free medium were much less pronounced and, in addition, no effect on the lipid composition was apparent. Conclusions: The comparison of the results indicates that bile action on surface properties is related to metabolic changes. Significance and Impact of the Study: Long-term exposure of bacteria to bile may cause metabolic changes affecting their adhesive properties irreversibly. This may be taken as a criterion to define the probiotic properties of different strains.