Objective: To investigate the efficacy and safety of using regional citrate anticoagulation (RCA) in the molecular adsorbent recirculating system (MARS) therapy for patients with acute-on-chronic liver failure (ACLF) grade 3. Methods: Twenty patients with ACLF grade 3 who were waiting for liver transplantation at our center from July 2018 to June 2019 were selected as the research participants. Clinical data from all patients were collected prior to MARS therapy, and all patients were treated with RCA for eight hours. Arterial blood gas analysis, total serum calcium, ionic calcium, and ionic calcium after filtration were collected at 0, 2, 4, and 6 hours of treatment and at the end of treatment. After filtration, the blood calcium was maintained within the range of 0.2-0.4 mmol/L, and the quantities of 4% sodium citrate solution and 5% calcium chloride solution were adjusted based on the monitored results. Changes in observed indices before and after treatment were compared, and the service life of the tubes and any therapeutic complications were recorded. Results: The 20 study patients received a total of 27 MARS treatments. Following treatment, the liver and renal function indices of the patients improved significantly (p < 0.05), with no serious disturbance of electrolytes or acid-base imbalance. A calcium ratio greater than 2.5 was detected on 10 occasions during MARS therapy, and the calcium ratio significantly increased at 2 hours following the start of MARS therapy (p < 0.05). As the therapeutic duration was extended, the calcium ratio gradually decreased. Of the 27 MARS treatments administered, a pipeline blockage occurred twice, and there was one episode of a severe disturbance of electrolytes caused by citrate accumulation leading to withdrawal of treatment. Conclusions: Patients with ACLF grade 3 may be safely treated with RCA as an effective anticoagulation treatment during MARS therapy.