Objective To understand the distribution and antimicrobial resistance of bacteria isolated from bile specimens in China in 2020-2024, and provide reference for clinical anti-infection treatment and rational antimicrobial use. Methods All data were collected from bacteria isolated from bile and antimicrobial susceptibility testing results reported by member units of China Antimicrobial Resistance Surveillance System (CARSS) in 2020-2024. Only the first strain of each patient was included. Constituent ratios and changing trends of antimicrobial resistance rates of bacterial strains were analyzed by Mantel-Haenszel χ² test. Results From 2020 to 2024, a total of 407 605 bacterial strains were included in the analysis. The top 4 isolated bacteria were Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis. Trend analysis showed that the detection rate of Klebsiella pneumoniae was on the rise (P<0.01), while those of Enterococcus faecium and Enterococcus faecalis were on the decline (both P<0.05). Antimicrobial susceptibility testing results showed that the resistance rates of major Gram-negative bacteria to piperacillin/tazobactam were generally on the rise, with that of Escherichia coli increasing from 8.7% to 14.3%. Detection rate of vancomycin-resistant Enterococcus faecium (VREM) increased from 0.4% to 1.7%, and that of carbapenem-resistant Escherichia coli (CRECO) increased from 2.3% to 2.6%. The resis-tance rates of major pathogenic bacteria to most third-generation cephalosporins presented decreasing trends, and the resistance rate of Escherichia coli to ceftazidime decreased from 34.2% to 29.8%. Compared with the overall data nationwide (taking 2023 as an example), the detection rates of CRECO and carbapenem-resistant Pseudomonas aeruginosa (CRPAE) from bile specimens were higher (2.4% vs 1.7%, 25.6% vs 16.3%, respectively), while the detection rates of carbapenem-resistant Acinetobacter baumannii (CRABA) and Klebsiella pneumoniae (CRKPN) were lower (40.4% vs 55.5%, 7.0% vs 10.8%, respectively). Conclusion The constituent of bacteria isolated from bile specimens in China from 2020 to 2024 is relatively stable, and the increase in VREM and CRECO detection rates is currently the main problem. Attention should be paid to the resis-tance of Pseudomonas aeruginosa isolated from bile specimens to carbapenems.