PeptidoglycanO-acetylation is a modification found in many bacteria. In Gram-positive pathogens, it contributes to virulence by conferring resistance to host lysozyme. However, in Gram-negative pathogens, its contribution to physiology and virulence is unknown. We examined the contribution ofpatA, patBandape1to peptidoglycanO-acetylation in the major human pathogenNeisseria meningitidis(Nm). Using genetic expression of all possible combinations of the three genes inEscherichia coliand Nm, we confirmed that PatA and PatB were required for PGO-acetylation, while ApeI removed theO-acetyl group. ApeI was active on allO-acetylated muropeptides produced by PatA and PatB during heterologous expression inE. coliand was also active on several PG structuresin vitro. Interestingly, in Nm, ApeI was found to preferentially de-O-acetylate muropeptides with tripeptide stems (GM3), suggesting that its activity is highly regulated. Accordingly, de-O-acetylation of GM3 regulated glycan chain elongation and cell size. Additionally, the virulence of Nm lacking ApeI was drastically reduced suggesting that regulation of glycan chain length byO-acetylation contributes to bacterial fitness in the host. Altogether, our results suggest that ApeI represents an attractive target for new drug development.