The uropathogenic Escherichia coli (UPEC) ST131 clone is of significant clinical importance due to its multidrug resistance phenotype and high prevalence worldwide. Genomic analyses have classified ST131 into three major clades (A, B and C), with all strains from the globally dominant clade C containing mutations in gyrA (L83S, N87D) and parC (I80S) that together confer high-level resistance to fluoroquinolones (FQs). Clade C strains can be further subdivided into clade C1 and C2, with clade C2 strains predominantly CTX-M-15-positive. The plasmid encoded enzyme Aac(6’)-Ib-cr confers reduced susceptibility to some FQs via acetylation of their piperazinyl group, and may prime the development of high-level resistance via mutations in gyrA and parC. Curiously, the presence of aac(6’)-Ib-cr gene is associated with clade C2 but not C1 strains (70.7% vs 3.7%), suggesting that gyrA and parC mutations were developed before the acquisition of aac(6’)-Ib-cr gene in clade C2. Here we sought to understand the role of the aac(6’)-Ib-cr gene in clade C2 strains highly resistant to FQs. We adapted a method utilizing transient suppression of DNA repair to perform gene editing on the chromosome of the reference clade C2 strain EC958 and reverted the gyrA and parC mutations (alone and in combination) back to E. coli K-12 FQ-sensitive. Using these revertants, we showed that Aac(6’)-Ib-cr conferred a 2-4 fold increase in MIC to ciprofloxacin in all strain backgrounds. We also showed that an EC958 mutant lacking aac(6’)-Ib-cr can be out-competed by EC958wt in human urine supplemented with ciprofloxacin (64 mg/L). Thus, the aac(6’)-Ib-cr gene may play an active role in the survival of C2 strains in urine of patients undergoing FQ treatment. This finding challenges the belief that the aac(6’)-Ib-cr gene is only important in FQ-sensitive strains, and suggests its acquisition contributes to the fitness of ST131 multidrug resistant clade C2 strains.