Objective To evaluate the efficacy and safety of in vitro anticoagulation with nafamostat mesilate in continuous renal replacement therapy (CRRT) in patients with sepsis complicated with acute kidney injury (AKI). Methods The study subjects were sepsis patients with AKI who underwent CRRT in West China Hospital of Sichuan University and were at high risk of bleeding. CRRT patients who received in vitro anticoagulation with nafamostat mesilate between July 2021 and January 2022 were included in the nafamostat group. The medical records of CRRT patients who did not use anticoagulants between January 2020 and December 2020 were retrospectively collected as a control group. The general situation, the lifespan of the first CRRT filter, the number of filters used within 72 hours of treatment, laboratory tests before and after treatment, and the occurrence of adverse reactions during treatment of the two groups of patients were analyzed. Results There were 42 patients in the control group and 21 patients in the nafamostat group. There was no statistically significant difference in age, gender, body mass index, mean arterial pressure, primary disease, Sequential Organ Failure Assessment score, Acute Physiology and Chronic Health Evaluation Ⅱ score, or pre-treatment laboratory test results between the two groups of patients (P>0.05). Kaplan-Meier survival analysis showed that the lifespan of the first filter was longer in the nafamostat group than in the control group (hazard ratio=0.408, P<0.05). The number of filters used by the control group patients after 72 hours of treatment was greater than that of the nafamostat group patients (2.1±0.6 vs. 1.3±0.5, P<0.05). After 72 hours of treatment, serum creatinine levels [(99.4±15.7) vs. (127.6±20.5)] μmol/L], urea nitrogen [(4.5±1.9) vs. (6.8±2.3) mmol/L], cystatin C [(1.0±0.2) vs. (1.2±0.2) mg/L], uric acid [(86.5±15.3) vs. (105.3±20.3) μmol/L] in the nafamostat group were lower than those of the control group (P<0.05), and there was no statistically significant difference in the results of other laboratory tests (P>0.05). There was no statistically significant difference in adverse reactions between the two groups of patients (P>0.05). Conclusion For patients with sepsis complicated with AKI who undergo CRRT and are at high risk of bleeding, nafamostat mesilate may be a safe and effective anticoagulant for in vitro anticoagulation.