Neisseria gonorrhoeae also called the gonococcus is the aetiological agent of the sexually transmitted disease gonorrhoea. The gonococcus is an obligate human pathogen and possesses the ability to survive intracellularly by the expression of virulence factors. Protein post-translational modifications are found in all organisms, and are involved in the regulation of the metabolism and gene transcription. In this study we investigated the role of non-enzymatic acetylation by acetyl-phosphate in N. gonorrhoeae. This was achieved through the deletion of the genes from the phosphotransacetylase-acetate kinase pathway (PTA-AKA) that control acetyl-phosphate production. As expected, high levels of protein acetylation were observed in the ΔackA strain. Using LC-MS/MS 59% of the N. gonorrhoeae proteome was identified, and we demonstrated that 48.3% of the proteome was acetylated. With many of the of the acetylated proteins being involved in central metabolism especially in pyruvate utilisation. Grow studies showed that the ΔackA strain was unable to utilise pyruvate as a carbon source, whereas it could grown on glucose as well as the wild-type. Furthermore, a deacetylase enzyme was identified and its gene mutated (Δhdac), this allowed the identification of a number of putative targets for HDAC, including phosphotransacetylase. We found that virulence was altered by the change of acetyl-phosphate concentration, with the ΔackA killing the wax moth larvae as a faster rate that the wild-type, whereas the Δpta strain was non-pathogenic in this model. The data obtained suggest that non-enzymatic protein acetylation in N. gonorrhoeae plays an important role in the central metabolism, carbon source utilisation and virulence of this bacterium.