Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.