The prevalence of fluoroquinolone (FQ)-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant (FQ^R) Campylobacter, it is necessary to understand the impact of FQ^R on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQ^RCampylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQ^RCampylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type (WT) allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant WT gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn), which are associated with FQ^R in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the WT gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQ^R mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the WT strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQ^RCampylobacter, modulates DNA supercoiling homeostasis in FQ^RCampylobacter.