OBJECTIVE: The aim of this study was to investigate the effects of halofuginone (HF) on gastric cancer cells and whether the combination of HF and trametinib has synergistic effects. INTRODUCTION: Halofuginone, a natural small molecule isolated from the plant , has been found to have anticancer activity in a variety of cancers, but few studies on HF in gastric cancer. METHODS: cell viability was performed using the CellTiterGlo assay and apoptosis and cell cycle analysis was performed by Annexin V-FITC staining and PI staining. We also analyzed RNA sequencing and differentially expressed genes was shown using the heatmap. Western blot and qPCR were carried out to determine the expression of pro-apoptotic and anti-apoptotic proteins. RESULTS: HF inhibited proliferation and induced apoptosis in gastric cancer cells in a dose-dependent manner. HF induced the expression of p-ERK in gastric cancer cells. HF and trametinib synergistically inhibited the gastric cancer cell proliferation. Trametinib inhibited HF-induced p-ERK expression. HF reduced anti-apoptotic protein Mcl-1 expression and reduced trametinib-induced upregulation of Mcl-1 expression. CONCLUSION: HF exerts its anti-cancer effects in gastric cancer and has a synergistic inhibition with trametinib, which may provide a novel therapeutic strategy for gastric cancer.