The balance between CD8 T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) plays a crucial role in the immune checkpoint inhibition (ICI) therapy in gastric carcinoma (GC). However, related factors leading to the disturbance of TME and resistance to ICI therapy remain unknown. In this study, we applied N6-methyladenosine (m6A) small RNA Epitranscriptomic Microarray and screened out 3'tRF-AlaAGC based on its highest differential expression level and lowest inter-group variance. N6-methyladenosine modification significantly enhanced the stability of 3'tRF-AlaAGC, which strengthened glycolysis and lactic acid (LA) production in GC cells by binding to PTBP1 (Polypyrimidine Tract Binding Protein 1). In the peritoneal GC implantation model established in huPBMC-NCG mice, 3'tRF-AlaAGC significantly increased the proportion of PD1 Treg cells. Furthermore, in high-LA environments driven by glucose consumption of GC cells, Treg cells actively uptake LA through MCT1, facilitating NFAT1 translocation into the nucleus and enhancing PD1 expression, whereas PD1 expression by effector T cell is diminished. Meanwhile, T cell suppression assays were performed under low-LA or high-LA conditions, and the proliferation of CD8 T cells was dampened by adding Sintilimab in a high-LA but not in a low-LA environment, suggesting the preferential activation of PD1 Treg cell. These findings deciphered the complexities of the immune microenvironment in GC, providing prospects for identifying robust biomarkers that could improve the evaluation of therapeutic effectiveness and prognosis in immune therapy for GC.