〔Abstract〕 Objective To investigate the expression, prognosis and biological role of hepatocyte nuclear factor 4A(HNF4A) in gastric cancer, and to study its effect on the proliferation of gastric cancer cells. Methods Tumor Immune Estimation Resource 2.0(TIMER2.0) and Gene Expression Profiling Interactive Analysis(GEPIA2) databases were used to analyze the relative expression levels of HNF4A in gastric cancer and normal tissue, KM Plotter was used to analyze the correlation between the expression level of HNF4A and the survival rate of gastric cancer patients, TISIDB database and R language(4.1.2) were used to analyze whether HNF4A was involved in the immune regulation process of gastric cancer. cBioPortal database was used to analyze the mutations of HNF4A in gastric cancer, GSEA 4.2 was used to analyze the functional enrichment of HNF4A, and LinkedOmics database was used to predict the genes that might be regulated by HNF4A. The relative expression of HNF4A in gastric cancer and adjacent tissues was detected by qRT-PCR, Western blot and immunohistochemistry(IHC). The proliferation and cell cycle of gastric cancer cells were analyzed by CCK-8, EdU, colony forming assay and flow cytometry. Results The expression of HNF4A increased in gastric cancer tissues(P<0.05), and the overall survival rate of gastric cancer patients with high HNF4A expression was worse(P<0.001). HNF4A was mainly missense mutated in gastric cancer. Immune cell infiltration showed that HNF4A was associated with B lymphocytes, CD8+T cells, neutrophils, macrophages and dendritic cells(allP<0.001). HNF4A was also associated with tumor mutation burden(r=0.28,P<0.0001) and microsatellite instability(r=0.13,P<0.01). After knockdown of HNF4A, cell proliferation ability was significantly inhibited, and cell cycle was arrested at G0/G1 phase. Conclusion HNF4A expression significantly increased in gastric cancer tissues, which is associated with poor prognosis, and may also be involved in immune regulation. Knockdown of HNF4A can inhibit the proliferation of gastric cancer cells.