Campylobacter jejuni is the leading cause of acute bacterial gastroenteritis in the developed world, however the biochemical mechanisms of pathogenesis remain poorly understood. Lysine acetylation (K-Ac) is a reversible post-translational modification that can alter in vivo protein structure / function. A combination of acetyl-lysine immunoprecipitation and two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified 5,569 acetylated lysines on 1,026 C. jejuni proteins (~63% of the predicted proteome). Functional enrichment confirmed acetylated proteins are involved in central metabolism, stress responses, translation and transcription, chemotaxis, gluconeogenesis and the TCA cycle. Label-based LC-MS/MS quantified acetylated peptides during growth in sodium deoxycholate (DOC, a component of gut bile salts), chicken exudate and mucin. A total of 1,445 proteins and 3,571 K-Ac peptides were quantified, and the most profound shift at the protein and acetylation level was as a result of DOC treatment. A total of 761 K-Ac peptides from 403 proteins were differentially regulated in response to DOC, the majority exhibiting an increase in abundance in K-Ac levels. These protein changes were involved in a number of metabolic pathways and processes, suggesting a dynamic role for K-Ac in bile resistance. We hypothesise a role for the acetogenesis/acetate utilisation pathway in protein acetylation and confirm K-Ac affects the stability of the major C. jejuni adhesin, CadF. This work provides the first system-wide interrogation and analysis of the lysine acetylome of C. jejuni and contributes to our understanding of acetylation as an emerging post-translational modification of interest in bacteria.