Objective ·To explore the regulatory mechanism of folate cycle metabolism in the immunosuppressive effect of myeloid derived suppressor cells (MDSCs). Methods ·Bone marrow cells were isolated from C57BL/6 mice and cultured in RPMI 1640 medium supplemented with GM-CSF, G-CSF, and IL-6 to induce MDSCs in vitro. PD-L1 expression level and ROS production level of induced MDSCs were detected by flow cytometry. CD8⁺ T cells were enriched from the spleen by MACS with anti-CD8a-conjugated microbeads, labeled with Celltrace violet, and then co-cultured with MDSCs. After 72 h, proliferation was assessed by flow cytometry. Folate cycle-related metabolic enzymes in MDSCs were detected by real-time quantitative PCR. MDSCs were treated with folate cycle metabolic enzyme MTHFD2 inhibitor DS18561882 (DS18) and folic acid antagonist Pemetrexed. ROS and mitoROS production in MDSCs were assessed by flow cytometry. CD8⁺ T cells were enriched from the spleen by MACS with anti-CD8a-conjugated microbeads, labeled with Celltrace violet, and then co-cultured with Pemetrexed or DS18-treated MDSCs. After 72 h, proliferation was assessed by flow cytometry. Transcript levels of folate cycle-related metabolic enzymes in pemetrexed or DS18-treated MDSCs were detected by RNAseq. A subcutaneous tumor mouse model of colon cancer was established. From the tenth day post-implantation, tumor-bearing mice were intraperitoneally injected with Pemetrexed (200 mg/kg) and tumor size was recorded for tumor growth curve. On the fourteenth day, mice were sacrificed, and tumors were harvested. MC38 tumor-bearing mice were treated with isotype antibody, anti-CD8 monoclonal antibody (1 mg/kg, deplete CD8⁺ T cells), Pemetrexed (200 mg/kg), and combination of Pemetrexed with anti-CD8 antibody. MC38 tumor-bearing mice were treated with isotype antibody, anti-Gr1 monoclonal antibody (1.25 mg/kg, clearing MDSCs), combination of Pemetrexed with anti-Gr1 antibody. On the tenth day after implantation, tumor-bearing mice were treated with Pemetrexed (50 mg/kg), anti-PD-1 monoclonal antibody (250 μg/kg), Pemetrexed, and combination of Pemetrexed with anti-PD-1 antibody. Results ·Flow cytometry data showed that PD-L1 level and ROS production were increased in induced MDSCs, and CD8⁺ T cell proliferation was also suppressed significantly. qPCR data revealed the expression of folate cycle-related metabolic enzymes MTHFD2 and others was increased in MDSCs. The accumulation of MDSCs was affected by DS18 or Pemetrexed, ROS production in MDSCs was reduced, and the immunosuppression of CD8⁺ T cells was relieved. RNA-seq results showed that genes related to MDSCs differentiation, such as S100 calc-binding protein A8, and genes related to MDSCs inhibition, such as cytochrome b-245β chain, which is related to ROS production, were also down-regulated after treatment with two folic acid cycling inhibitors. Tumor growth was suppressed by Pemetrexed. Tumor progression was promoted by combination of Pemetrexed with anti-CD8 antibody, compared with Pemetrexed monotherapy. However, tumor growth delay was inhibited by combination of Pemetrexed and anti-CD8, compared with anti-CD8 monotherapy. Tumor growth delay was caused by MDSCs depletion. But tumor growth was promoted by combination of pemetrexed and anti-Gr1, compared with pemetrexed monotherapy. Tumor growth was restricted by combination of pemetrexed and anti-PD-1 antibody, compared with anti-PD-1 monotherapy. Conclusion ·Pemetrexed relies on CD8⁺ T cells for anti-tumor effects and further retards tumor growth by reprogramming MDSCs to an anti-tumor phenotype. Modulating MDSCs by targeting folate cycle could impair their immunosuppressive ability and enhance the efficacy of immune checkpoint blockade in cancer treatment.