To investigate the clinical efficacy and safety of polymyxin B in the treatment of sepsis caused by extensively-drug resistant (XDR) Gram-negative bacteria. A retrospective analysis of 39 septic patients with XDR Gram-negative bacterial infection treated with polymyxin B in the department of critical care medicine of Xiangya Hospital of Central South University from June 2018 to September 2019 were enrolled. The clinical characteristics, bacterial culture, the sensitivity antibacterial drugs, types and courses of antibiotics, biochemical indexes, and acute physiology and chronic health evaluation II (APACHE II) before and after polymyxin B treatment were collected, to assess microbial clearance and efficacy, drug related adverse effects, and 28-day mortality in septic patients with XDR. Of the 39 septic patients with XDR, 32 (82.1%) were male, with the mean age of (53.6±12.6) years old. The main infection site was pulmonary infection (51.2%), and the treatment courses of polymyxin B were ≥ 5 days. A total of 66 pathogenic bacteria were detected from 39 patients. Among them, with the high estrate of detecting Acinetobacter baumannii of 51.5% (34/66). After treatment with polymyxin B, the results showed that the clearance rate of microorganisms was 65.2% (43/66), the overall effective rate was 59.0% (23/39), and the 28-day all-cause mortality was 41.0% (16/39). There were no significant differences in clinical efficacy and microbial clearance among patients with different treatment groups of polymyxin B [< 10 days, 10-15 days, and > 15 days groups: effective rates were 56.5% (13/23), 54.5% (6/11), 80.0% (4/5), χ 2 = 0.999, P = 0.728; the microbial clearance rates were 43.5% (10/23), 54.5% (6/11), and 80.0% (4/5), χ 2 = 2.141, P = 0.393]. The effective and microbial clearance rates of the polymyxin B daily doses of 150 mg and 200 mg groups were significantly higher than those of the daily dose of 100 mg [effectiveness: 85.7% (6/7), 87.5% (7/8) vs. 41.7% (10/24); microbial clearance rate: 71.4% (5/7), 87.5% (7/8) vs. 33.3% (8/24), all P < 0.05], however, there were no significant differences in the length of intensive care unit (ICU) stay and mechanical ventilation time among different daily dose groups. The APACHE II score after polymyxin B administration was significantly lower than before administration (all patients: 16.20±9.24 vs. 24.40±4.73, effective patients: 11.30±4.08 vs. 23.00±4.56, both P < 0.05). Four patients with renal injury had an increase in serum creatinine during the administration of polymyxin B, and recovered after discontinuation of the drug without other adverse reactions. Polymyxin B can be used as an effective treatment option for patients with severe infection of XDR Gram-negative bacteria.