Tetrahydrofolate and its derivatives, collectively termed folates or vitamin B9, are essential cofactors for one-carbon metabolism. They transport and donate Cl-units for the synthesis of pantothenate, purines, thymidylate, serine, glycine, methionine and formylmethionyl-tRNA. Also, recent studies indicate that folates can act as electron donors in major cellular processes. Plants and many microorganisms synthesize folates de novo through a complex metabolic route that is now fully elucidated. In contrast, humans and other vertebrates lack a complete biosynthetic pathway and thus need dietary folates, of which plants are major sources. Folate deficiency is widespread in rich and developing countries and is associated with severe health problems. Supplementation of foods with synthetic folic acid and biofortification is an alternative strategy to fight folate deficiency. Encouraging pilot metabolic engineering studies in plants enabled significant enhancement of folate contents. In the next future, increasing our knowledge about the mechanisms controlling folates homeostasis in plants will provide the keys towards efficient biofortification of plant foods.