Although it has been shown that polymorphisms in one-carbon metabolism (OCM) pathway are associated with gastric cancer (GC), their interactions and contributions for patients' survival are elusive. In this study, we investigated the effects of polymorphisms and their interactions on the survival of GC patients, including genes of Methylenetetrahydrofolate reductase (MTHFR 677C > T, 1298A > C), Methionine synthase reductase (MTRR 66A > G), Methionine synthase (MTR 2756A > G), and Thymidylate synthase (TS 3'-UTR ins6 > del6, 5'-UTR 2R > 3R). We recruited 919 GC patients from 1998 to 2006. The Kaplan-Meier plots, Cox regression analyses and the log-rank tests were carried out in this study. MTHFR 1298CC genotype showed protective effect (HR = 0.444, 95% CI = 0.210-0.940). MTRR 66 GA + GG genotypes decreased the risk of death (HR = 0.793, 95% CI = 0.651-0.967) in general, and in subgroups with more pronounced diffuse type, greater depth of invasion (T2/T3/T4), higher level lymph node metastasis (N1/N2/N3), advanced TNM stages (II/III level) and 5-Fu treatment. However, the improved survival disappeared when GC patients simultaneously had MTR 2756 GA + GG genotypes (HR = 1.063, 95% CI = 0.750-1.507). Although MTRR 66GA genotype was not associated with the survival of GC patients, patients with simultaneous MTRR 66GA and MTR 2756AA genotypes exhibited significant risk reduction of death (HR = 0.773, 95% CI = 0.609-0.981). MTHFR 1298 CA + CC combined with TS 5-UTR 2R3R + 3R3R genotypes (HR = 0.536, 95% CI = 0.315-0.913) also increased patient survival rates. Our results suggest that the MTRR 66A > G and MTHFR 1298A > C polymorphisms may be useful prognostic biomarkers for GC patients.