Helicobacter hepaticus is an enterohepatic bacterium associated with inflammatory bowel disease in children and causes severe hepatobiliary disorders in mice. To elucidate the molecular response of H. hepaticus to bovine bile, a proteomic investigation was conducted. Bacteria were grown for 48 h in liquid media supplemented with different concentrations of bovine bile to determine its effects on bacterial growth and morphology. Protein expression profiles of bacteria grown at a bile concentration of 0.1% and in the absence of bile were obtained using two-dimensional gel electrophoresis. Gel spots with differences in intensities greater than 2-fold between both conditions were determined, and 55 differentially expressed proteins were identified using tandem mass spectrometry. Identified proteins participate in various biological functions including cell envelope biosynthesis, cell response to stress, iron homeostasis and transport, motility, primary and secondary metabolism, and virulence. Changes in the expression of H. hepaticus genes related to proteins involved in virulence and oxidative stress that were differentially expressed in the presence of bile were investigated using real-time reverse transcriptase PCR. The results indicated that the effects of bile on H. hepaticus included a strong response to oxidative stress and an expression of factors that can promote host colonization.