Background: In recent years, New Delhi metallo-beta-lactamases 1 (bla(NDM-1)) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the blaNDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China. Methods: Antimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids. Results: Ten E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6')-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with similar to 33.3 kb size that carried the bla(NDM-1) gene, except T3 strain, of which the bla(NDM-1) gene was located on a similar to 50 kb plasmid. Conclusions: Our findings suggested that plasmid was able to contribute to the dissemination of blaNDM-1. Hence, more attention should be devoted to monitor the dissemination of the bla(NDM-1) gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.