ABSTRACT. Folic acid and methylenetetrahydrofolate reductase (MTHFR) may both affect the development of human cancer. We conducted a population-based case-control study in a Chinese population to investigate the potential role of folate intake and MTHFR gene polymorphisms in gastric cancer, and their interaction with infection by Helicobacter pylori and tumor location. A total of 767 patients with newly diagnosed gastric cancer and 775 controls were selected for this study. Genotyping of MTHFR C677T and A1298C was conducted by TaqMan assays using the ABI Prism 7911HT Sequence Detection System, and information 9719 MTHFR and folate and gastric cancer risk ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (4): 9718-9726 (2014) on folate intake was collected by questionnaire. Compared with the CC genotype of MTHFR C677T, the TT genotype was significantly associated with a decreased risk of gastric cancer when the analysis was adjusted for other potential risk factors. We found a marginal significantly decreased risk of gastric cancer for individuals carrying the T allele [adjusted odds ratio (OR) = 0.83; 95% confidence interval (CI) = 0.65-1.01]. We detected an inverse relationship between folate intake and risk of gastric cancer, and the adjusted ORs (95%CI) for moderate and high folate intake were 0.97 (0.74-1.25) and 0.64 (0.49-0.87), respectively. Moreover, H. pylori infection, folate intake, and location of the tumor showed a significant interaction with the MTHFR C677T polymorphism. Our study suggests a protective role of MTHFR 677TT and high folate intake against gastric cancer, and the effect of the MTHFR C677T genotype may differ by H. pylori infection, folate consumption, and tumor site.