Objective To explore the clinical significance and antimicrobial resistance of group B Streptococcus (GBS) isolated from midstream urine culture, aiming to provide a basis for the diagnosis and treatment of clinical urinary tract infection (UTI). Methods Information about GBS strains isolated from midstream urine culture of inpatients and outpatient in a hospital in Nanjing from February 2020 to December 2022 were retrieved through laboratory information system, strains with complete data were screened out. Case data, urine routine, and antimicrobial susceptibility testing results were reviewed. Results A total of 9 081 non-repetitive bacterial strains were detected from midstream urine culture, including 425 GBS strains, accounting for 4.7%, ranking sixth. Strains with incomplete data were excluded, a total of 365 patients were included in the study. 169 (46.3%) were males and 196 (53.7%) were females, with an average age of (55.4±15.2) years. 365 patients who were detected GBS were from 17 departments, with the highest proportion being department of urology (n=237, 64.9%). The underlying diseases of patients mainly included hypertension (n=136), diabetes(n=95), urolithiasis (n=120) and urinary tumors (n=98). 211 patients underwent urological surgery, all were treated with antimicrobial agents before surgery, and 205 patients underwent indwelling urinary catheters after surgery; 9 patients were detected GBS from urine during the middle and advanced stage of pregnancy. 36.4% (n=133), 38.9% (n=142) and 24.7%(n=90)patients had GBS colony count ≤ 10⁴ CFU/mL, 10⁴-10⁵ CFU/mL, and ≥ 10⁵ CFU/mL, respectively. Patients with symptoms of UTI accounted for 24.9% (n=91), and asymptomatic bacteriuria accounted for 75.1% (n=274). The incidence of UTI symptoms in males was lower than that in females (19.5% vs 29.6%, P < 0.05). As the GBS colony count in urine culture increased, the proportion of patients with symptoms of UTI showed an upward trend (P < 0.05). On the day of urine culture, the positive rates of urine routine white blood cells, leukocyte esterase, and nitrite were 53.2%, 50.1%, and 3.8%, respectively. The positive rates of urine occult blood, leukocyte esterase, white blood cells, and urine protein in patients with symptomatic UTI were all higher than those with asymptomatic bacteriuria patients (all P < 0.05). No GBS were found to be resistant to penicillin, ampicillin, vancomycin, linezolid, and tigecycline. The resistance rate to levofloxacin and moxifloxacin was about 40%, and resistance rate to tetracycline and clindamycin was over 60%. Conclusion GBS isolated from urine is more common in non-pregnant adults, and only a small percentage have symptoms of UTI. The results of urine culture and urine routine should be comprehensively judged based on patient's clinical symptoms and signs. GBS in urine is susceptible to multiple antimicrobial agents, and clinical medication should be adopted rationally based on antimicrobial susceptibility testing result.