Escherichia coli ST131 is a cause for global concern because of its high multidrug resistance and several virulence factors. In this study, the contribution of acrAB-TolC efflux system of E. coli ST131 to fluoroquinolone resistance was evaluated. A total of nonrepetitive 111 ciprofloxacin-resistant E. coli isolates were included in the study. Multilocus sequence typing was used for genotyping. Expressions of acrA, acrB, and TolC efflux pump genes were measured by RT-PCR. Mutations in marA, gyrA, parC, and aac(6')-lb-cr positivity were studied by Sanger sequencing. Sixty-four (57.7%) of the isolates were classified as ST131, and 52 (81.3%) of the ST131 isolates belonged to H30-Rx subclone. In ST131, CTX-M 15 positivity (73%) and aac(6')-lb-cr carriage (75%) were significantly higher than those in non-ST131 (12.8% and 51%, respectively) (P < 0.05). The ampicillin-sulbactam (83%) resistance was higher, and gentamicin resistance (20%) was lower in ST131 than that in non-ST131 (64% and 55%, respectively) (P = 0.001 and P = 0.0002). Numbers of the isolates with MDR or XDR profiles did not differ in both groups. Multiple in-dels (up to 16) were recorded in all quinolone-resistant isolates. However, marA gene was more overexpressed in ST131 compared to that in non-ST131 (median 5.98 vs. 3.99; P = 0.0007). Belonging to H30-Rx subclone, isolation site, ciprofloxacin MIC values did not correlate with efflux pump expressions. In conclusion, the marA regulatory gene of AcrAB-TolC efflux pump system has a significant impact on quinolone resistance and progression to MDR profile in ST131 clone. Efflux pump inhibitors might be alternative drugs for the treatment of infections caused by E. coli ST131 if used synergistically in combination with antibiotics.