Microsporidians are a diverse group of obligate intracellular parasites that have minimized their genome content and simplified their sub-cellular structures by reductive evolution. Functional studies are limited because we lack reliable genetic tools for their manipulation. Here, we demonstrate that the cristae-deficient mitochondrion (mitosome) of the microsporidian Trachipleistophora hominis is the functional site of iron-sulphur cluster (ISC) assembly, which we suggest is the essential task of this organelle. Cell fractionation, fluorescence imaging and fine-scale immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified recombinant mitosomal ISC proteins. Reconstitution proceeded as rapidly and efficiently as observed for yeast or fungal mitochondrial ISC components. Core components of the T. hominis cytosolic iron-sulphur protein assembly (CIA) pathway were also identified including the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that both the ISC and CIA biosynthetic pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of the Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides additional compelling evidence for the ancient chimeric ancestry of eukaryotes.