Campylobacter jejuni is a pathogen that colonizes the intestinal tract of humans and some animals. The in vitro responses of the bacterium to ox-bile were studied using proteomics to understand the molecular mechanisms employed by C. jejuni to survive bile stress. Its in vitro tolerance to bile was determined by growing the bacterium for 18 h in liquid cultures containing different bile concentrations. Significant growth inhibition was observed in the presence of 2.5% bile, and a decrease of 1.12 log units was measured at a bile concentration of 5%. Protein expression profiles of bacteria grown with and without bile were compared using two-dimensional polyacrylamide gel electrophoresis. Proteins with differential intensities greater than two-fold were identified using tandem mass spectrometry. Nuclear magnetic resonance spectroscopy and spectrophotometry were employed to measure enzyme activities in cell extracts from bacteria grown with and without bile. Together with proteins known to be involved in C. jejuni bile tolerance, the presence of bile modulated the expression of proteins such as elongation factors, ferritin, chaperones, ATP synthase and others, previously unknown to be implicated in the response of the bacterium to bile.