Fruits and certain vegetables have a protective effect on gastric cancer (GC) and folate is one of the nutrients in fruits and vegetables. We hypothesized that the polymorphisms of thymidylate synthase (TYMS) gene involved in folate metabolism are associated with GC risk. In a population-based case-control study of 337 GC cases and 326 controls, frequency-matched by age, sex and residential areas in a southern Chinese population, we genotyped the 28 bp tandem repeat in the TYMS 5'-untranslated enhanced region (TSER) and the 6 bp deletion/insertion at bp 1494 in the TYMS 3'-untranslated region (TS3'UTR). We found that although the TSER polymorphism had no main effect on GC risk, the TS3'UTR 6 bp/6 bp genotype was associated with a significantly increased risk of GC [adjusted odds ratio (OR) = 1.96, 95% confidence interval (CI) = 1.18-3.25], especially the non-cardiac gastric cancer (2.16, 1.22-3.82), compared with the 0 bp/0 bp genotype. However, when we evaluated these two polymorphisms together and used the combined genotype with zero variant allele (TSER 2R and TS3'UTR 6 bp variant alleles) as the reference, we found that the combined genotype with three or four variant alleles was associated with a significantly increased risk of GC (2.06, 1.12-3.79), especially the non-cardiac gastric cancer (2.33, 1.19-4.59), and this significant association was more pronounced among older women (>60 years old), non-smokers, and never tea drinkers. In conclusion, the TYMS polymorphisms, especially the TS3'UTR polymorphism, are associated with GC risk, especially the non-cardiac gastric cancer, and the TSER 2R and TS3'UTR 6 bp alleles may jointly play a role in the etiology of GC in the southern Chinese population. Larger studies are warranted to verify these findings.