Enrofloxacin treatment of chickens infected with fluoroquinolone(FQ)-sensitive Campylobacter promoted the emergence of FQ-resistant Campylobacter mutants which propagated in the intestinal tract and recolonized the chickens. The recovered isolates were highly resistant to quinolone antibiotics but remained susceptible to non-FQ antimicrobial agents. Specific single-point mutations in the gyrA gene and the function of the CmeABC efflux pump were linked to the acquired FQ resistance. These results reveal that Campylobacter is hypermutable in vivo under the selection pressure of FQ and highlight the need for the prudent use of FQ antibiotics.