Objective <p>Gastric cancer (GC) is a highly invasive malignancy with a propensity for lymph node metastasis. This study investigated how lactylation of TRIM29 contributes to the invasive behavior of GC and lymph node metastasis and the efficacy of chemotherapy for the disease.</p> Methods <p>We examined the expression levels of TRIM29 and its lactylation status in GC tissues and cell lines using quantitative reverse-transcription polymerase chain reaction, immunohistochemistry based on tissue microarrays and western blotting. Functional transwell migration, three-dimensional invasion assay and tube formation assays were performed to assess the role of TRIM29 in GC. The interaction between TRIM29 and heteronuclear ribonucleoprotein A1(hnRNPA1) was explored by co-immunoprecipitation and mass spectrometry.</p> Results <p>Expression of TRIM29 was significantly upregulated in GC tissues in comparison with adjacent non-tumor tissues. This upregulation was associated with lymph node metastasis, vascular tumors and a worse prognosis. Lactylation of TRIM29 in GC cells enhanced the migratory ability and invasiveness of these cells and lymph node metastasis. Mechanistically, TRIM29 formed a complex with hnRNPA1, which in turn activated the Wnt/β-catenin signaling pathway by stabilizing β-catenin in a ubiquitination-dependent manner. Targeting TRIM29 and lymphangiogenesis augmented the efficacy of 5-fluorouracil-based chemotherapy.</p> Conclusion <p>Lactylation of TRIM29 promotes invasive behavior and lymph node metastasis in GC cells by engaging the hnRNPA1-mediated Wnt/β-catenin pathway. Targeting TRIM29 and lymphangiogenesis may be a promising therapeutic strategy for patients with advanced GC.</p>