Abstract: Objective To describe the molecular characteristics and antibiotic resistance of Vibrio cholerae O139 in Shanghai from 2005 to 2014. Methods PCR was conducted to detect six virulence-related genes of 86 strains of V. cholerae O139, including cholera toxin gene (ctxA), hemolysin gene (hlyA)、toxin-coregulated pilus gene (tcpA), the regulatory protein genes (toxR) and the genes of phage CTX encoding cholera toxin (zot, ace). Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out for the molecular subtyping of the strains, and the strains' antibiotic resistance was detected with K-B test. Results Up to 93.2% of V. cholerae O139 strains isolated from patients and 58.3% isolated from aquatic products were toxigenic,while all the V. cholerae O139 strains isolated from environment were non-toxigenic. According to the PFGE pattern, 86 V. cholerae O139 strains belonged to 35 molecular types. According to MLST result, 86 V. cholerae O139 strains belonged to 9 ST types. ST 69 and ST 162 were predominant in Shanghai. The toxigenic V. cholerae O139 strains isolated showed higher resistance to antibiotics compared with the non-toxigenic strains. Conclusion Most V. cholerae O139 strains isolated from patients and aquatic products in Shanghai were toxigenic, PFGE suggested that aquatic products were the main infectious sources of cholera. The toxigenic isolates had similar molecular characteristics and the predominant type was ST 69. The antibiotic resistance of the toxigenic V. cholerae O139 strains was serious.