The ability of Helicobacter pylori to tolerate bile is likely to be important for its colonization and survival in the gastrointestinal tract of humans. As bile can be acidified after reflux into the low pH of the human stomach, the inhibitory effect of fresh human bile with normal appearance on H. pylori before and after acidification was tested first. The results showed that acidification of bile attenuated its inhibitory activity towards H. pylori. Next, the protein profiles of H. pylori under human bile and acidified bile stress were obtained by two-dimensional electrophoresis. Protein spots with differential expression were identified using tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that the changes in proteomic profiles under bile and acidified bile stress were similar when compared with that of normal H. pylori. Expression of 28 proteins was found to be modulated, with the majority being induced during bile or acidified bile exposure. These proteins included molecular chaperones, proteins involved in iron storage, chemotaxis protein, enzymes related to energy metabolism and flagellar protein. These results indicate that H. pylori responds to bile and acidified bile stress through multiple mechanisms involving many signalling pathways.