Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMetto fMet-tRNAfMetis crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, usingin vivoandin vitroapproaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in thein vitroassay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmtstrain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment toEscherichia colishowed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.