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The emergence of ceftriaxone-resistant Neisseria gonorrhoeae is a current public health concern for several countries. To explore the potential genes related to ceftriaxone resistance in N. gonorrhoeae, we subcultured six gonococcal strains on increasing concentrations of ceftriaxone and isolated one that became resistant. After analyzing several genes known to be involved in ceftriaxone resistance, we found a single mutation in penA (A501V). However, differential analysis of the genomes and transcriptomes between pre- and post-selection strains revealed many other mutated genes, as well as up-and down-regulated genes. Transformation of mutated gene penA into non-resistant strains increased the MICs between 2.0 and 5.3 fold, and transformation of mutated ftsX increased MICs between 3.3 and 13.3 fold. Genes encoding ABC transporters, FarB, Tfq, Hfq, and ExbB were overexpressed while pilM,N, and Q were down-regulated. Furthermore, the resistant strain produced cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and down regulated PilMNQ. In conclusion, antimicrobial pressure could result in emergence of ceftriaxone resistance, and the evolvement of Neisseria gonorrhoeae resistance to ceftriaxone is a complicated process at both pre-transcriptional and post-transcriptional levels involving several resistance mechanisms of increased efflux and decreased entry.