Objective To analyze the distribution and changing trend of antimicrobial resistance of bacteria isolated from bile from Hunan Province Antimicrobial Resistance Surveillance System. Methods Data of pathogens isolated from bile from Hunan Province Antimicrobial Resistance Surveillance System from 2012 to 2021 were collected. The constituent of bacteria and antimicrobial susceptibility testing results were analyzed by WHONET 5.6 software. Changes in antimicrobial resistance was analyze by trend chi-square test. Results The major pathogenic bacteria isolated from bile were Gram-negative bacteria, accounting for 70.84%. The top three isolated Gram-negative pathogens were Escherichia coli (30.14%), Klebsiella pneumoniae(12.15%), and Pseudomonas aeruginosa(5.18%), and the top two Gram-positive bacteria were Enterococcus faecium (10.34%)and Enterococcus faecalis(9.52%). The resistance rates of Klebsiella pneumoniae and Escherichia coli to imipenem were highest in 2012-2013, being 15.7% and 14.9%, respectively, presenting an downward trend (P<0.05); resistance rates to piperacillin/tazobactam and cefoperazone/sulbactam were <24%, presenting an upward trend year by year (P<0.05); the susceptibility rate to amikacin was >94%, to levofloxacin and ciprofloxacin was 15.5%-65.2%. The highest resis-tance rate of Pseudomonas aeruginosa to imipenem (32.0%) was higher than that of meropenem (22.9%), resis-tance rates to piperacillin/tazobactam and cefoperazone/sulbactam were <19%. The highest resistance rates of Acinetobacter baumannii to imipenem and meropenem were 59.4% and 62.6%, respectively, resistance rate to cefo-perazone/sulbactam was <48%, presenting an upward trend (P<0.05); the highest resistance rate to ciprofloxacin (60.8%) was higher than levofloxacin (48.7%); resistance rate to tigecycline was <8%. The resistance rates of Enterococcus faecium to penicillin and ampicillin were both higher than those of Enterococcus faecalis, presenting an upward trend (P<0.05). Resistance rate of Enterococcus faecium to vancomycin was lower than that of Enterococcus faecalis. The resistance rates of Enterococcus faecium to vancomycin and linezolid were 0.5%-4.5% and 0.5%-3.4%, respectively; resistance rates of Enterococcus faecalis to vancomycin and linezolid were 0.2%-1.7% and 0.5%-3.5%, respectively (both P<0.05), all presenting a downward trend (all P<0.05). Conclusion Pathogenic bacteria isolated from bile are mainly related to the intestinal flora. The resistance rates of Enterococcus faecalis and Enterococcus faecium to vancomycin and linezolid as well as resistance rate of Enterobacterales to carbapenem antibiotics all present a downward trend.