Background:MitoNEET is a mammalian iron-sulfur protein with the ability to transfer iron-sulfur (Fe-S) in vitro. Results: MitoNEET conveys Fe-S from the mitochondrion to the cytosol and reactivates cytosolic iron regulatory protein 1 into an Fe-S aconitase. Conclusion: A novel mitoNEET-dependent Fe-S repair pathway affects a key regulator of iron metabolism. Significance: MitoNEET is the first mitochondrial protein found to be involved in mammalian cytosolic Fe-S repair.