Background: Emergence of multidrug-resistant Neisseria gonorrhoeae resulting from new genetic mutations is a serious threat to controlling gonorrhea. Goal: To determine 1) antimicrobial susceptibilities and the corresponding genetic mutations and 2) the role of MtrRCDE efflux system in gonococcal resistance to fluoroquinolones. Study Design: Antimicrobial susceptibility testing and sequence analysis of gyrA, parC, and mtrR loci of 131 N. gonorrhoeae isolates from Japan. Results: The proportion of N. gonorrhoeae strains resistant and intermediate-resistant to antimicrobials was 25.2% and 48.9% for ciprofloxacin, 25.2% and 30.5% for ofloxacin, 12.2% and 53.4% for penicillin; and 17.6% and 51.1% for tetracycline, respectively. Strains were categorized into 22 mutation profiles, with GyrA-S91F/ParC-D86N/MtrR-G45D being the most predominant profile. The frequency of mutation in gyrA, parC, mtrR, and the mtrR promoter was 71%, 47.3%, 77.1%, and 23.7%, respectively. Seventy-one percent of strains carried mutations in both gyrA and mtrR. Conclusion: This study reports simultaneous mutations in fluoroquinolone target enzymes and the MtrRCDE efflux system as a fluoroquinolone-resistant mechanism in N. gonorrhoeae.