Abstract: Objective To understand the antibiotic resistance of frequently isolated clinical gram negative strains and provide evidence for the treatment of clinical infections caused by these bacilli. Methods A total of 2361 frequently isolated gram negative bacilli strains isolated in our hospital from January 2009 to December 2011 were analyzed retrospectively. Of the 2361 isolates, 1832 were Enterobacteriaceae strains, including 1053 Escherichia coli strains, 504 Klebsiella pneumoniae strains, 102 Enterobacter cloacae strains, 74 Enterobacter aerogenes strains, 46 Serratia marcescens strains, 28 Morganella morganii strains and 25 Klebsiella oxytoca strains, 529 were Nonfermentative bacilli strains, including 272 Acinetobacter baumannii strains, 148 Pseudomonas aeruginosa strains and 109 Burkholderia cepacia strains. Results The resistance rates of Enterobacteriaceae strains to impenem and meropenem were as follows: E. coli:0.2% (2/1053) and 0.4% (4/1053); K. pneumoniae:3.0% (15/504) and 3.4% (17/504); E. cloacae: 2.9% (3/102) and 3.9% (4/102); E. aerogenes: 9.5% (7/74) and 8.1% (6/74), S. marcescens: 21.7% (10/46) and 19.6% (9/46); M. morganii: 10.7% (3/28) and 14.3% (4/28); K. oxytoca: 16.0% (4/25) and 16.0% (4/25), respectively. While the resistance rates of A. baumannii, P. aeruginosa and B. cepacia to imipenem and meropenem were 76.8% (209/272) and 73.9% (201/272), 37.2% (55/148) and 33.8% (50/148), and 73.4% (80/109) and 69.7% (76/109), respectively. The resistance rates of tested clinical isolates to other 10 antibiotics were very high. Conclusion Carbapenems have relatively high antimicrobial activity to Enterobacteriaceae in vitro, while low antimicrobial activity to nonfermentative bacilli strains.