Folate metabolism supports the synthesis of nucleotides as well as the transfer of methyl groups. Polymorphisms in folate-metabolizing enzymes have been shown to affect risk of colorectal neoplasia and other malignancies. Using data from a population-based incident case-control study (1,600 cases and 1,962 controls), we investigated associations between genetic variants in the reduced folate carrier (RFC), thymidylate synthase (TS), methionine synthase (MTR), and 5,10-methylenetetrahydrofolate reductase (MTHFR) and colon cancer risk. The TS enhancer region (TSER) variant was associated with a reduced risk among men [2rpt/2rpt versus 3rpt/3rpt wild-type; odds ratio (OR), 0.7; 95% confidence interval, 0.6-0.98] but not women. When combined genotypes for both TS polymorphisms (TSER and 3'-untranslated region 1494delTTAAAG) were evaluated, ORs for variant genotypes were generally below 1.0, with statistically significantly reduced risks among women. Neither MTR D919G nor RFC 80G>A polymorphisms were associated with altered colon cancer risk. Because folate metabolism is characterized by interrelated reactions, we evaluated gene-gene interactions. Genotypes resulting in reduced MTHFR activity in conjunction with low TS expression were associated with a reduced risk of colon cancer. When dietary intakes were taken into account, individuals with at least one variant TSER allele (3rpt/2rpt or 2rpt/2rpt) were at reduced risk in the presence of a low folate intake. This study supports findings from adenoma studies indicating that purine synthesis may be a relevant biological mechanism linking folate metabolism to colon cancer risk. A pathway-based approach to data analysis is needed to help discern the independent and combined effects of dietary intakes and genetic variability in folate metabolism.