Zinc finger protein 154 (ZNF154) has been identified as a tumor suppressor gene in multiple carcinomas. Lymph node (LN) metastasis is one of the most intensively negative factor of gastric cancer (GC) prognosis. However, the potential mechanisms of ZNF154-mediated LN metastasis are not elucidated. This study aimed to investigate the role of ZNF154 in LN metastasis of GC and their underlying mechanisms through in vitro and in vivo experiments.Antitumor effect was measured by growth inhibition by cell counting kit-8 (CCK-8) and colony formation assay. Cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell migration and invasion were measured by wound healing and transwell invasion assays, respectively. The expression levels of proteins were analyzed by Western blot. Xenograft models were used for validation in vivo.Our research showed that ZNF154 was down-regulated in 81.43% (57 of 70) of GC tissues compared with 58.6% of paired non-tumor tissues from patients, ZNF154 was down-regulated in 100% (7 of 7) of GC cell lines, up-regulated expression of ZNF154 in MGC-803 GC cells reduced cell proliferation, viability, migration and invasion, and enhanced cell apoptosis and arrested cell cycle in G2 phase, and suppressed tumorigenicity of MGC-803 cells in mice. Furthermore, up-regulated expression of ZNF154 mRNA reduced the expression of B-cell lymphoma-2 (Bcl-2), matrix metalloproteinase 2 (MMP-1), hepatocyte growth factor (HGF), vascular endothelial growth factor-A/C (VEGF-A/C).ZNF154 inhibited LN metastasis of GC cells by suppressing several biological events of GC cells. ZNF154 was a tumor suppressor gene that is a promising target for blocking nodal involvement in GC.© 2022 He et al.