Background: ABO-incompatible liver transplantation (ABO-i LT) has become a rescue therapeutic option for severe hepatic failure patients. Although the use of rituximab greatly reduces the morbidity of antibody-mediated rejection (AMR), severe adverse effects, such as infection and biliary complications still seriously threatening the survival of the transplant recipients. The aim of this study is to evaluate the safety and feasibility of using mesenchymal stem cells (MSCs) to replace rituximab in ABO-i LT. Methods: 22 severe hepatic failure patients undergoing ABO-i LT were enrolled and randomly divided into two groups, including the MSC group and Rituximab group. The safety of the application of MSCs and the incidence of allograft rejection, including antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were evaluated in both groups at 2-year follow-up period as primary endpoints. Recipients and graft survivals and other postoperative complications were compared as secondary endpoints. Results: No severe adverse effects occurred in the MSC group related to the MSC infusion. MSCs treatment obtained comparable, if not better, results to rituximab for decreasing the incidence of acute rejection, especially AMR (9.1% vs 27.3%). Inspiringly, compared to the Rituximab group, biliary complications (0% vs 45.5%) and infection (9.1% vs 81.8%), were significantly decreased in MSCs group. In addition, there were no significant difference in 2-year graft and recipient survivals in the two groups (81.8% vs 72.7%). Conclusions: Our data shows that MSCs transfusion is comparable to rituximab treatment for AMR prophylaxis following ABO-i LT. Additionally, the results indicate that MSC is more beneficial to the prevention of infection and biliary complications, which may be introduced as a novel immunosuppressive approach for ABO-i LT. Trial registration: Trial registration: chictr.org.cn, ChiCTR2000037732. Registered 31 August 2020- Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=57074.