Objective To understand the distribution and antimicrobial resistance of bacteria from clinical urine specimens in Hunan Province, and provide scientific basis for anti-infection treatment of patients with urinary tract infection (UTI). Methods Bacterial resistance surveillance was carried out according to the technical scheme requirements of China Antimicrobial Resistance Surveillance System, antimicrobial susceptibility testing results were judged based on standard of American Clinical and Laboratory Standards Institute, data about strains and antimicrobial resistance of bacteria from urine specimens reported by member units of Hunan Province Antimicrobial Resistance Surveillance System in 2012-2021 were analyzed with WHONET 5.6 software. Results A total of 379 330 strains of bacteria were isolated from urine specimens in 2012-2021, Gram-negative and Gram-positive bacteria accounted for 75.3% (72.4%-76.0%) and 24.7% (24.0%-27.6%), respectively. The top 5 Gram-negative bacteria were Escherichia coli (48.3%), Klebsiella pneumoniae (8.4%), Proteus mirabilis (3.3%), Pseudomonas aeruginosa (3.0%) and Enterobacter cloacae (1.6%). The top 5 Gram-positive bacteria were Enterococcus faecium (8.1%), Enterococcus faecalis (6.6%), Staphylococcus epidermidis (1.9%), Staphylococcus aureus (1.9%) and Staphylococcus haemolyticus (1.4%). The constituent of bacteria isolated from urine specimens of patients in different gender and age groups were different. Escherichia coli ranked first, accounting for 34.8% and 57.2% in males and females, respectively, as well as 49.0% and 34.4% in adults and children, respectively. Escherichia coli maintained high susceptibility to carbapenems, amikacin, tigecycline, cefoperazone/sulbactam and furantoin, with resistance rate < 10%, while resistance to cefazolin, cefuroxime, ceftriaxone, cefotaxime and quinolones were relatively higher, with resistance rate >48%; resistance rates to ceftazidime, ceftriaxone, cefotaxime, cefepime and aztreonam presented decreased trend (all P < 0.001). Klebsiella pneumoniae maintained higher susceptibility to carbapenems, amikacin and tigecycline, with resistance rate < 11%, resistance rate to quinolones was much lower than that of Escherichia coli. Enterococcus faecium and Enterococcus faecalis maintained high susceptibility to vancomycin, teicoplanin and linezolid, with resistance rates < 5%; resistance rate of Enterococcus faecalis to ampicillin and furantoin was < 15%. Except for linezolid and minocycline, resistance rates of Enterococcus faecium to the other tested antimicrobial agents were all higher than Enterococcus faecalis. No Staphylococcus aureus was found to be resistant to vancomycin, linezolid and teicoplanin. Conclusion Escherichia coli is the main bacteria isolated from urine specimens from various member units of Hunan Province Antimicrobial Resistance Surveillance System. In early empirical treatment, clinical antimicrobial should be targetedly used as early as possible based on bacterial identification and antimicrobial susceptibility testing results, so as to improve treatment effectiveness and slow down the emergence of antimicrobial resistance.