BACKGROUND Gastric cancer is one of the most common and deadly malignancies worldwide. Despite recent medical progress, the 5-year survival rate of gastric cancer is still unsatisfactory. 5-fluorouracil (5-Fu) is one of the first-line antineoplastic treatments for gastric cancer, as it can effectively induce cancer cell apoptosis. However, the effect of 5-Fu is limited due to drug resistance of the malignant tumor. Previous studies have reported that Sotetsuflavone from Cycas revoluta Thunb. can markedly suppress lung cancer cell proliferation by apoptosis, though its effect on gastric cancer remains unknown. AIM To investigate the inhibitory effect of Cycas revoluta Thunb. and to determine whether it can overcome gastric cancer cell drug resistance to 5-Fu. METHODS Cell viability was examined to determine whether the natural extract of Cycas revoluta Thunb. induced gastric cancer cell death. The half-maximal effective concentration and the half-maximal lethal concentration were calculatede. Wound-healing and transwell assays were performed to examine gastric cancer cell motility. Clonogenic assays were performed to investigate the synergistic effects of Cycas revoluta Thunb. with 5-Fu, and apoptotic bodies were detected by Hoechst staining. Western blotting was performed to examine the expression of related proteins and to investigate the molecular mechanism of Cycas revoluta Thunb.-induced cancer cell apoptosis. The expressions of proteins, including mammalian target of rapamycin (mTOR) and p-AKT, were detected in different combinations of treatments for 48 h, then analyzed by ECL detection. RESULTS Gastric cancer cells were more sensitive to the natural extract of Cycas revoluta Thunb. compared to normal gastric epithelial cells, and the extract effectively inhibited gastric cancer cell migration and invasion. The extract improved the anti-cancer effect of 5-Fu by enhancing the chemosensitization of gastric cancer cells. Extract plus 5-Fu further reduced the expression of the drug-resistance-related proteins p-AKT and mTOR after 48 h compared to 5-Fu alone. Compared to 5-Fu treatment alone, mTOR and p-AKT expression was significantly reduced by about 50% and 75%, respectively. We also found that the natural extract of Cycas revoluta Thunb. further increased 5-Fu-induced gastric cancer cell apoptosis. Expression of apoptosis-related protein X-linked inhibitor of apoptosis protein and apoptosis inducing factor were significantly reduced and increased, respectively, in the 5-Fu-resistant gastric cancer line SGC-7901/R treated with extract plus 5-Fu, while the expression of survivin did not change. CONCLUSION The natural extract of Cycas revoluta Thunb. effectively inhibited gastric cancer cell growth and enhanced the anti-cancer effect of 5-Fu through the AKT-mTOR pathway.