Campylobacter jejuni is the leading cause of acute bacterial gastroenteritis in the developed world. The biochemical mechanism of pathogenesis however, remain poorly understood. Lysine acetylation (KAc) is a reversible post-translational modification that may alter target protein 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 key metabolic and virulence-associated pathways. Label-based LC-MS/MS quantified the unmodified proteome and KAc sites associated with growth in sodium deoxycholate (DOC, a component of gut bile salts). A total of 1,445 proteins and 3,571 KAc peptides were quantified. Of these, 761 acetylated peptides from 403 proteins were altered in abundance following growth in DOC, the majority exhibiting an increase in KAc. Protein changes were involved in metabolic pathways and processes, suggesting a dynamic and regulatory role for KAc in bile resistance. As with other enteric bacteria, we confirmed that KAc was largely chemically mediated by acetyl-phosphate (AcP), rather than via protein acetyltransferases, and this process involves the acetogenesis / acetate utilisation pathway and the sole bacterial deacetylase CobB that regulates acetyl-CoA synthetase (Acs). Unlike other bacteria, we noted several surface-associated virulence factors that displayed enrichment of multiple KAc sites. The major C. jejuni adhesin CadF was acetylated at 10 sites, several of which occurred proximal to known proteolytic cleavage sites that confer immune avoidance during infection. A reduction in KAc on these sites was shown to correlate with increased CadF processing, suggesting KAc may confer protection from proteases during periplasmic translocation and contribute to regulation of in vivo virulence. CobB inhibition did not attenuate global increases in DOC-associated KAc and had no influence on any acetylated sites in CadF. This work provides the first system-wide analysis of the lysine acetylome in C. jejuni and contributes to our understanding of KAc as an emerging modification in bacteria.