Frataxin homologues are important iron chaperones in eukarya and prokarya. Using a native proteomics approach we were able to identify the structural frataxin homologue Fra (formerly YdhG) of Bacillus subtilis and to quantify its native iron-binding stoichiometry. Using recombinant proteins we could show in vitro that Fra is able to transfer iron onto the B. subtilis SUF system for iron–sulfur cluster biosynthesis. In a four-constituents reconstitution system (including SufU, SufS, Fra and CitB) we observed a Fra-dependent formation of a [4 Fe–4 S] cluster on SufU that could be efficiently transferred onto the target apo-aconitase (CitB). A Δfra deletion mutant showed a severe growth phenotype associated with a broadly disturbed iron homeostasis; this indicates that Fra is a central component of intracellular iron channeling in B. subtilis.