Human tumors are generally methionine (MET)-dependent in that their growth is inhibited by MET-depletion down to levels that will still allow normal cell growth. The differential effect of methionine depletion on tumor and normal cells has suggested that methionine depletion may be able to modulate many and possibly all classes of cancer drugs. In this report, we determined if MET-depletion could modulate 5-fluorouracil (5-FU) efficacy on the human gastric cancer xenograft, SC-1-NU in nude mice. The tumor-bearing mice were treated with a MET-free diet and intraperitoneal administration of 5-FU at a dose of 30 mg/kg given for four cycles. MET depletion enhanced the antitumor activity of 5-FU by approximately two-fold with statistical significance of p < 0.05. The MET-free diet increased intratumoral thymidylate synthetase inhibition early after 5-FU administration; Therefore, MET-depletion was thought to increase the 5-FU antitumor activity by modulating intratumoral folate metabolism. The data in this report suggest the high clinical potential of methionine depletion, combined with 5-FU and leucovorin on refractory tumors such as stomach cancer.