Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to every antimicrobial used against it. Ceftriaxone-resistant N. gonorrhoeae strains harbor mosaic penA alleles that encode highly mutated forms of Penicillin-Binding Protein 2 (PBP2), the lethal target of ceftriaxone. When bacteria develop resistance to antibiotics through an extensive alteration of an essential protein, they often incur a fitness cost. To have success as a pathogen, they must overcome this fitness deficit. We have hypothesized that antibiotic-resistant clinical isolates acquire compensatory mutations to help alleviate the fitness cost associated with resistance. Wild-type strains harboring a mosaic penA allele have reduced fitness both in vitro and in vivo, but compensatory mutants with increased biological fitness were isolated. In one such strain, a mutation (G348D) in acnB, which encodes the tricarboxylic acid (TCA) cycle aconitase, was identified. The hallmark of this compensatory mutant is that it grows rapidly during log-phase growth, but then plateaus early as it enters into stationary phase growth. The acnB-G348D mutation is both necessary and sufficient to impart the growth phenotype characteristic of compensatory mutant. Western blots of AcnB during the growth cycle show that wild-type AcnB increases in abundance as the cells enter stationary phase, whereas in the AcnB mutant, expression decreases by 2- to 3- fold. Interestingly, acnB mRNA levels do not account for changes in AcnB protein levels. These data suggest that acnBG348D acts as a functional knockout in regards to the role of AcnB in the TCA cycle. Any fitness benefits associated with this mutation do not appear to be directly associated with the activity of AcnB in the TCA cycle.