Abstract Background: A relationship between dietary folate intake and efficacy of fluorouracil (FU) is supported by preclinical data. Furthermore, there are several reports that evaluated genetic polymorphisms of MTHFR (methylenetetrahydrofolate reductase) or TYMS (thymidylate synthase) and efficacy of FU. However, to our knowledge, there are no reports that evaluate simultaneously the effects of folate intake and genetic polymorphisms on clinical outcome of gastric cancer patients. Methods: We retrospectively analyzed the survival impact of estimated folate intake by a food frequency questionnaire and MTHFR and TYMS polymorphisms in 132 patients with advanced gastric cancer who were treated with first-line FU-based chemotherapy. Results: Median overall survival was 11.3 months (95% confidence interval, 9.4-13.4 mo) and median progression-free survival was 5.2 months (95% confidence interval, 4.1-6.3 mo). Patients with folate intake of >260 μg/day (n = 88) showed longer overall survival compared with low folate intake (n = 44; overall survival, 12.2 versus 8.4 mo). In a multivariate Cox model, patients who had folate intake of >260 μg/day, MTHFR 677 TT polymorphism, and TYMS-3′ untranslated region 6-bp insertion were associated with better survival. Similar tendency was observed in progression-free survival. No interaction was observed between folate intake and favorable genotypes. Conclusion: Folate intake and genetic polymorphisms of MTHFR and TYMS were associated with better clinical outcome by FU-based chemotherapy in advanced gastric cancer. Impact: Our results suggested folate intake and folate-related genetic polymorphisms may play an important role in efficacy of FU-based chemotherapy in advanced gastric cancer. Cancer Epidemiol Biomarkers Prev; 19(5); 1311–9. ©2010 AACR.