Lactobacillus rhamnosus GG (GG) is a widely used and intensively studied probiotic bacterium. Although the health benefits of strain GG are well documented, the systematic exploration of mechanisms by which this strain exerts probiotic effects in the host has only recently been initiated. The ability to survive the harsh conditions of the gastrointestinal tract, including gastric juice containing bile salts, is one of the vital characteristics that enables a probiotic bacterium to transiently colonize the host. Here we used gene expression profiling at the transcriptome and proteome levels to investigate the cellular response of strain GG toward bile under defined bioreactor conditions. The analyses revealed that in response to growth of strain GG in the presence of 0.2% ox gall the transcript levels of 316 genes changed significantly (p < 0.01, t test), and 42 proteins, including both intracellular and surface-exposed proteins (i.e. surfome), were differentially abundant (p < 0.01, t test in total proteome analysis; p < 0.05, t test in surfome analysis). Protein abundance changes correlated with transcriptome level changes for 14 of these proteins. The identified proteins suggest diverse and specific changes in general stress responses as well as in cell envelope-related functions, including in pathways affecting fatty acid composition, cell surface charge, and thickness of the exopolysaccharide layer. These changes are likely to strengthen the cell envelope against bile-induced stress and signal the GG cells of gut entrance. Notably, the surfome analyses demonstrated significant reduction in the abundance of a protein catalyzing the synthesis of exopolysaccharides, whereas a protein dedicated for active removal of bile compounds from the cells was up-regulated. These findings suggest a role for these proteins in facilitating the well founded interaction of strain GG with the host mucus in the presence of sublethal doses of bile. The significance of these findings in terms of the functionality of a probiotic bacterium is discussed.