• The addition of Hexokinase 2(HK2)inhibitor 3-Bromopyruvic acid (3-BrPA) into traditional myeloid-derived suppressor cells (MDSCs) induction system in vitro promoted MDSCs production. • Treatment of 3-BrPA increased the expression of MDSC-related immunosuppressive molecules. • 3-BrPA enhances the ability of MDSCs to suppress T cells in mixed lymphocyte cultivate. • 3-BrPA enhances the immunosuppressive effect of MDSCs via STAT3/HK2 pathway. • 3-BrPA-treated MDSCs significantly prolonged the survival time of mouse heart allografts. The adoptive transfer of ex vivo generated myeloid-derived suppressor cells (MDSCs) may be a promising therapeutic strategy for preventing allograft rejection after solid organ transplantation. Currently, the precise role of immune-metabolic pathways in the differentiation and function of MDSCs is not fully understood. Hexokinase 2 (HK2) is an isoform of hexokinase and is a key enzyme involved in the increased aerobic glycolysis of different immune cells during their activation and function. Here, we demonstrate that the addition of HK2 inhibitor 3-Bromopyruvic acid (3-BrPA) into traditional MDSCs induction system in vitro significantly promoted MDSCs production and enhanced their immunosuppressive function. Treatment with 3-BrPA increased the expression of MDSC-related immunosuppressive molecules, such as iNOS, Arg1, and CXCR2. Moreover, the adoptive transfer of 3-BrPA-treated MDSCs significantly prolonged the survival time of mouse heart allografts. This study provides a novel strategy to solve the problems of harvesting enough autologous cells for MDSC production from sick patients, and producing functionally enhanced MDSCs for preventing graft rejection and inducing tolerance. [ABSTRACT FROM AUTHOR]