ObjectiveTo investigate the distribution and antimicrobial resistance of infection caused by clinically isolated Enterobacteriaceae from a hospital．MethodsData about Enterobacteriaceae isolated from January 2005 to June 2012 was analyzed statistically．Results A total of 5 538 isolates of Enterobacteriaceae were isolated, the top three specimen sources were urine（35.63%，1 973 isolates），respiratory tract（32.38%，1 793 isolates），wound secretion, drainage and pus（10.88%，603 isolates）； the top five isolated pathogens were Escherichia coli （49.41%，2 736 isolates）, Klebsiella pneumoniae （25.30%，1 401 isolates）, Enterobacter cloacae（6.37%，353 isolates）, Proteus mirabilis（4.24%，235 isolates） and Salmonella spp. （4.17%，231 isolates）. The resistant rates of most Enterobacteriaceae to imipenem and meropenem were ＜2%; amikacin and piperacillin/tazobactam were with high activity against Enterobacteriaceae, the resistant rate was ＜10%. Resistance rate of Escherichia coli from sputum specimens was significantly higher than those from blood and urine specimens（both P＜0.05）.ConclusionEnterobacteriaceae in this hospital are mainly isolated from urine and respiratory tract, and with varied degree of resistance to different antimicrobial agents, carbapenems remains high activity against Enterobacteriaceae，attention should be paid to the emergence of carbapenemresistanct strains．