Introduction Dual antimicrobial therapy with ceftriaxone plus azithrothmycin has recently been recommended for uncomplicated gonorrhoea in the United States, the United Kingdom and Canada because of increases in MICs of N. gonorrhoeae for extended-spectrum cephalosporins. However, emergence of high-level azithromycin resistance in N. gonorrhoeae have been reported in many countries. No high-level azithromycin-resistant isolates of N.gonorrhoeae have been reported in China. Azithromycin has been added into antibiotic susceptibility panel since 2013. In this study, we present the results of amtimicrobial susceptibility testing of 384 gonococcal strains isolated between 2013 and 2014 and evidence of high-level azithromycin resistance in Nanjing, China. Methods 384 N. gonorrhoeae isolates were isolated sequentially from male adults with symptoms/signs of urethritis attending a single STD clinic in Nanjing, China between 2013 and 2014. Minimum inhibitory concentrations (MICs) of N. gonorrhoeae to penicillin, tetracycline, ciprofloxacin, spectinomycin, azithromycin, cefixime and ceftriaxone were determined by the agar dilution technique. b-lactamase production was determined by paper acidometric testing. Results Resistance to penicillin and tetracycline was 72.1% (277/384) and 85.9% (330/384), respectively; 46.9% (180/384) of strains were PPNG and 34.6% (133/384) were TRNG. All isolates (100%) were resistant to ciprofloxacin (MIC ≥1 μg/ml). A total of 32.3% (124/384) of isolates were resistant to azithromycin (MIC ≥1μg/ml), among them 25% (31/124) isolates displayed high-level azithromycin resistance (MIC ≥256 μg/ml). All isolates were susceptible to spectinomycin, cefixime and ceftriaxone. However, 16 isolates (4.3%) had elevated MICs (≥0.125 μg/ml) for cefixime and 38 isolates (10.1%) had an MIC of 0.125 μg/ml for ceftriaxone. Conclusion The present study shows a high prevalence of N. gonorrhoeae isolates displaying resistance to penicillin, tetracycline, azithromycin and ciprofloxacin and reduced susceptibility to extended-spectrum cephalosporins. High-level azithromycin resistance in N. gonorrhoeae has emerged in Nanjing, China. Disclosure of interest statement Nothing to declare