ObjectiveTo investigate the distribution and antimicrobial resistance of pathogens from bile specimens from patients with biliary tract infection, and provide reference for clinical antimicrobial use.MethodsBile specimens collected from May 2011 to May 2013 were cultured, identification and antimicrobial susceptibility testing of the isolated bacteria were performed by VITEK2 COMPACT automatic system, antimicrobial susceptibility data were analyzed using the WHONET 5.3 software.ResultsOf 445 cultured bile specimens, 254 (57.08%) were positive for bacterial culture. A total of 306 pathogenic isolates were obtained, 231 (75.49%) of which were gramnegative strains，58 (18.95%) were grampositive strains, and 17(5.56%) were fungi. The most common gramnegative pathogens in biliary tract infection were Escherichia coli(21.89%), Klebsiella pneumoniae(18.30%)，and Pseudomonas aeruginosa (13.40%); the most common grampositive pathogens were Enterococcus faecalis (5.56％)，coagulase negative Stahylococcus (3.59％),and Enterococcus faecium (3.59％)．Antimicrobial resistant rate of gramnegative bacteria to imipenem and ertapenem was the lowest, followed by cefotetan, amikacin, and piperacillin /tazobactam;the resistant rates to quinolones,penicillins,and cephalosporins were high. Antimicrobial resistant rate of grampositive bacteria to vancomycin was the lowest, followed by linezolid, the resistant rates to other antimicrobial agents were 30%-90%.ConclusionThe major pathogens in biliary tract infection are gramnegative bacilli, regular monitor on antimicrobial resistance is important for the treatment.