Cofactor F plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence， antibiotic biosynthesis， pro-drug activation and methanogenesis. However， the biosynthetic pathway for F has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate， nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that， along with its archaeal homolog CofC， it accepts phosphoenolpyruvate， rather than 2-phospho-L-lactate， as the substrate， leading to the formation of the previously uncharacterized intermediate dehydro-F-0. The C-terminal domain of FbiB then utilizes FMNH to reduce dehydro-F-0， which produces mature F species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F from a recombinant F biosynthetic pathway in Escherichia coli.