Abstract: Objective: To observe the inhibitory mechanism of agrimoiin on human gastric cancer cell line HGC-27 and MKN-45. Methods: Gastric cancer HGC-27 and MKN-45 cells were divided into control group, different doses of agrimoniin groups, capecitabine (positive control) group and agrimoniin+NRF2 activator (Bardoxolone) group. Cell counting kit-8 (CCK-8) assay was used to detect the viability of HGC-27 and MKN-45 cells. Scratch test was used to observe the cell migration ability. Flow cytometry was used to detect the level of intracellular reactive oxygen species (ROS). Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of malondialdehyde (MDA), glutathione (GSH) and oxidized glutathione (GSSG). Western blotting was used to detect the expression of PI3K-AKT pathway-related proteins and anti-ferroptosis molecules NRF2, Histone H3, HO-1, SLC7A11 and GPX4. Results: Compared with the control group, different doses of agrimoniin had a significant inhibitory effect on the activity of HGC-27 and MKN-45 cells (P＜0.05), and the inhibitory effect increased with the extension of time and the increase of agrimoniin dose. Compared with the control group, the migration ability of HGC-27 and MKN-45 cells in the low-dose and high-dose agrimoniin groups and the positive control group was weakened, the levels of ROS and MDA were increased, the ratios of GSH/GSSG, P-PI3K/PI3K, and P-AKT/AKT were decreased, and the expression of Total-NRF2, nuclear-NRF2, HO-1, SLC7A11, and GPX4 proteins was down-regulated (all P＜0.05). The protein expression levels of Total-NRF2 and GPX4 were increased in the agrimoniin +Bardoxolone group (P＜0.05). Conclusion: Agrimoniin can promote ferroptosis in gastric cancer HGC-27 and MKN-45 cells, and its mechanism may be related to inhibiting the activation of PI3K-AKTNRF2 signaling pathway.