Background &amp; Aims <p>The role of histone lysine lactylation (Kla) in gastric cancer mesenchymal stromal cells (GCMSCs) remains elusive. This study aimed to investigate the potential lactyltransferase and the influence of histone Kla in GCMSCs.</p> Methods <p>Histone Kla levels in GCMSCs were characterized by cell immunofluorescence and Western blotting. The transcriptional regulation was validated via CUT&amp;Tag sequencing coupled with qPCR analysis. Immunosuppressive effectors through Western blotting and multiplex immunohistochemistry (mIHC). β-alanine and AZD3965 were applied to inhibit histone Kla in vitro. Ultimately, β-alanine was selected for its ability to enhance the efficacy of anti-PD-1 therapy in NCG^PBMC tumor-bearing model.</p> Results <p>The primary GCMSCs exhibited higher histone Kla levels. Lactate treatment triggered the nuclear translocation of aminoacyl-tRNA synthetase 1 (AARS1), whose knockdown attenuated lactate-driven nuclear accumulation and suppressed histone Kla. CUT&amp;Tag-qPCR analysis revealed lactate-induced Pan-Kla enrichment at the <i>FAP</i> promoter, driving extracellular matrix (ECM) receptor interaction pathways. Deeply, H4K8 lactylation (H4K8la) has been defined as the key regulatory role to promote <i>FAP</i> transcription, corroborated by mIHC mapping of their immune-excluded spatial organization. In NCG^PBMC tumor-bearing models, β-alanine synergized with anti-PD-1 therapy, enhancing tumor regression and augmenting CD8⁺ T cell effector functions.</p> Conclusions <p>We elucidate a Kla-dependent mechanism underlying GCMSCs-mediated ECM remodeling and immunosuppressive niche formation. The results provide novel insights into the epigenetic regulation of immunosuppressive TME.</p> Graphical abstract <p>H4K8 lactylation promotesFAP mediated ECM remodeling and CD8⁺ T cell exclusion.Combining a lactylation inhibitor with anti PD 1 enhances CD8⁺ T cell infiltration andtumor suppression.</p>