Abstract: OBJECTIVE To investigate the distribution and drug susceptibility of pathogens cultured from bile specimens of patients with acute cholangitis so as to provide guidance for reasonable use of antibiotics. METHODS A total of 145 patients with acute cholangitis who were treated in hospitals from Jun 2009 to Jun 2015 were enrolled in the study, then the bile specimens were collected from the patients, the bacterial identification was carried out by using WITEK-2 Compact, the drug susceptibility testing was conducted by means of K-B disk diffusion method, and the statistical analysis of data was performed with the use of SPSS15.0. RESULTS Totally 158 strains of pathogens were isolated from 195 submitted bile specimens that were obtained from 145 patients, of which 58 (36.71%) were gram-positive bacteria, 95 (60.13%) were gram-negative bacteria, and 5 (3.16%) were fungi. The drug susceptibility rates of the gram-positive bacteria to teicoplanin, imipenem, and vancomycin were more than 75.00%, while the drug susceptibility rates to penicillin G, azithromycin, and ofloxacin were low. The drug susceptibility rates of the gram-negative bacteria to piperacillin, meropenem, imipenem, and amikacin were more than 75.00%, while the drug susceptibility rates to ampicillin, cefotaxime, and aztreonam were low. The drug susceptibility rates of the fungi to ketoconazole, fluconazole, itraconazole, and voriconazole were 100.00%. CONCLUSION The gram-negative bacteria are dominant among the pathogens causing the infections in the patients with cholangitis. The drug susceptibility rates of the gram-positive bacteria and gram-negative bacteria to the commonly used antibiotics are decreased to a certain degree, and it is necessary for the hospital to reasonably use antibiotics based on the results of the drug susceptibility testing.