The present invention provides a 5-methylfolate producing microorganism which a) has been modified to have a decreased expression and/or activity of a polypeptide having both dihydrofolate synthase activity and folylpolyglutamate synthetase activity compared to an otherwise identical microorganism (reference microorganism); b) has been (further) modified to express a heterologous polypeptide having only dihydrofolate synthase activity; c) has been (further) modified to have an increased expression level of at least one enzyme (such as at least two, at least three, at least four, at least five, at least six, at least seven or at least eight) enzymes involved in the biosynthesis of a 5-methylfolate compared to an otherwise identical microorganism (reference microorganism); and/or d) has been (further) modified to have a decreased expression and/or activity of a polypeptide having 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase activity compared to an otherwise identical microorganism (reference microorganism).