There is growing evidence that folate metabolism is involved in migraine pathophysiology, mainly in migraine with aura. Even though folate metabolism is regulated by a number of enzymes, only two functional polymorphisms have been tested in association studies with migraine. Here, we have explored the possible role in migraine of other folate-metabolizing enzymes which are in close interdependency with 5',10'-methylenetetrahydrofolate reductase analyzing functional polymorphisms of these enzymes in a case-control study. Individually, thymidylate synthase (TS), methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthase (MTHFD1), or methionine synthase (MS) polymorphisms did not modify the general risk for suffering migraine. Nevertheless, we observed a strong interaction between TS and MTHFR mutated genotypes, which increased over 8-fold the risk for experiencing aura among migraineurs; MTHFD1 and MTHFR mutated genotypes also increased together the risk for migraine in general (OR = 3.08; 95% CI = 1.3-7.4). We conclude that the pathogenetic role of the MTHFR T677 allele in migraine is modulated by functional polymorphisms of TS and MTHFD1.