Abstract: Objective To investigate the trend of specimen source，distribution and antimicrobial agents resistance of Enterobacteriaceae from 2012 to 2014. Methods Enterobacteriaceae isolated from a affiliated hospital of a Medical College were identified by BD Phoenix.and the data were judged with CLSI2013 and analyzed with WHONET5.6. Results A total of 9 031 strains of Enterobacteriaceae were collected,and mainly come from the ICU,neurology,neurosurgery,urology,and respiratory department，and mainly come from specimens of sputum,urine,blood.Secretion,ascites and cerebrospinal fluid.The strains mainly included Klebsiella pneumoniae,Escherichia coli,Proteus mirabilis,Enterobacter cloacae,Serratia marcescens and Enterobacter aerogenes.The strains showed high sensitivity ratio for carbapenems,enzyme inhibitor and amino glycosides. Conclusion Enterobacteriaceaes were mainly pathogen in the hospital,and expressed high resistance for most antibiotics. Carbapenems were still the optimum selection for control infections of Enterobacteriaceae,but Carbapenem-Resistant Enterobacteriaceae(CRE) already appeared increasingly.Clinicians should pay attention to CRE and promote the rational use of antimicrobial agents to avoid the spread of CRE in hospital.