Abstract: OBJECTIVE To investigate the distribution and drug resistance spectrum of clinical gram-negative bacterial isolates in our hospital during 2006-2007 for the guidance of rational use of antibiotics. METHODS Clinical isolates were collected from clinical departments. Drug sensitivity tests were carried out using disc diffusion test, and were analyzed by WHONET 5.4. RESULTS Among 3775 Gram-negative bacterial isolates collected in two years. While 36.3% were Non-fermentative bacteria. The major isolates in the 3775 strains were Escherichia coli, Klebsiella pneumoniae, Haemophilus parainfluenzae, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia. The detection rates of ESBLs to Escherichia coli and Klebsiella pneumonia were 62.7% and 42.6%. The activity of imipenem and meropenem against Enterobacteriaceae was excellent sensitive (>94%), while Haemophilus influenzae and Haemophilus parainfluenzae were sensitive to cefuroxime or ceftriaxone（>89%）. Acinetobacter baumannii showed the drug-resistant rate below 8.8% to imipenem, meropenem, cefoperazone and minocycline. The most active agent against Pseudomonas aerugionsa was tobramycin, followed by meropenem, ceftazidime, amikacin and imipenem (10.1%-16.1%).Minocycline was the most active agent to Stenotrophomonas maltophilia, Chryseobacterium meningosepticum,Chryseobacterium indologen, and Burkholderia cepacia (1.3%-8%). CONCLUSION Carbapenems remained very high activity against Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa.Using the antibiotics reasonably according to the type of pathogenic bacteria and the result of drug sensitivity is effective on infection, and this is an important tool to reduce the form of drug-resistant strains.