Objective To investigate the distribution and resistance of Gram-negative bacteria in intensive care unit, so as to help physicians to choose reasonable antibiotics. Methods Case histories were collected in which laboratory findings of Gram-negative bacteria in the intensive care unit in hospital during 2013 were recorded. A statistical analysis was made of the basic conditions of patients ,the sorts of pathogenic bacteria, the results of AST and bacterial drug resistance. Results In all the samples examined, there were 461 strains in all, in which there were 328 strains of gram-negative bacteria ,consisting of 71.1%.In all the gram-negative bacteria separated, there were mainly Acinetobacter baumannii and Pseudomonas aeruginosa, the ratio being 48.2% and 23.5%. 82.9% of Acinetobacter baumannii and 94.8% of Pseudomonas aeruginosa were separated from sputum, respectively. Imipenem shows great multiantibiotic resistance to Acinetobacter baumannii and stenotrophomonas maltophilia, and it is sensitive to the other gram-negative bacteria. Stenotrophomonas maltophilia is sensitive to quinolone antimicrobial agents, such as levofloxacin and ciprofloxacin. While Acinetobacter baumannii shows highly multiantibiotic resistance to antibiotics except amikacin and polymyxin E. Conclusions In the 2013, the separation rate of gram-negative bacteria in the ICU in this hospital was very high. And the infection of gram-negative in the intensive care unit was severe. It is important to carry out surveillance of bacteria resistance and the growth of bacteria resistance in time, which can guide the doctor to use drugs reasonably.