ABSTRACTTherapeutic options are limited forNeisseria gonorrhoeaeinfection, especially for oral drugs. The purpose of this study was to investigate the susceptibility ofN. gonorrhoeaeto oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains ofN. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59N. gonorrhoeaestrains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P< 0.0001). However, a mutation of codon 40 (Ala→Asp) in themtrRgene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P< 0.0001). InN. gonorrhoeaemultiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in themtrRpromoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance inN. gonorrhoeaeinfection.