BackgroundThe appropriate administration regimen of polymyxin B is yet controversial. The present study aimed to explore the optimal dose of polymyxin B under therapeutic drug monitoring (TDM) guidance.MethodsIn China’s Henan province, 26 hospitals participated in a randomized controlled trial. We included patients with sepsis caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) susceptible to polymyxin B. The patients were randomly divided into a high-dose (HD) group or a low-dose (LD) group and received 150 mg loading dose, 75 mg every 12 h and 100 mg loading dose, 50 mg every 12 h, respectively. TDM was employed to determine if the dose of polymyxin B needs adjustment based on the area under the concentration–time curve across 24 h at a steady state (ssAUC0–24) of 50–100 mg h/L. The primary outcome was the 14-day clinical response, and the secondary outcomes included 28- and 14-day mortality.ResultsThis trial included 311 patients, with 152 assigned to the HD group and 159 assigned to the LD group. Intention-to-treat analysis showed that the 14-day clinical response was non-significant (p= 0.527): 95/152 (62.5%) in the HD group and 95/159 (59.7%) in the LD group. Kaplan–Meier’s 180-day survival curve showed survival advantage in the HD group than in the LD group (p= 0.037). More patients achieved the target ssAUC0–24in the HD than in the LD group (63.8% vs. 38.9%;p= 0.005) and in the septic shock subgroup compared to all subjects (HD group: 71.4% vs. 63.8%,p= 0.037; LD group: 58.3% vs. 38.9%,p= 0.0005). Also, the target AUC compliance was not correlated with clinical outcomes but with acute kidney injury (AKI) (p= 0.019). Adverse events did not differ between the HD and LD groups.ConclusionA fixed polymyxin B loading dose of 150 mg and a maintenance dose of 75 mg every 12 h was safe for patients with sepsis caused by CR-GNB and improves long-term survival. The increased AUC was associated with increased incidence of AKI, and TDM results were valued to prevent AKI.Trial registrationTrial registration ClinicalTrials.gov: ChiCTR2100043208, Registration date: January 26, 2021.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13054-023-04522-6.