Mak16 and its interacting partner Rpf1 play a critical role at an early step in the maturation of the ribosomal 60S subunit, as revealed by recent cryo-EM structures.1 While most studies suggested no metal participation or the presence of a Zn2+ ion in Mak16, we identify a bona fide Fe/S cluster in yeast Mak16 through both in vivo and in vitro methods. We demonstrate a functional link between the mitochondrial and cytosolic Fe/S protein biogenesis and ribosome assembly, highlighting a previously unexplored aspect of 60S ribosomal biogenesis. Interestingly, 17 % of all Mak16 proteins, including those from pathogens like Trypanosoma and Leishmania, have a C-terminal tryptophan residue as likely CIA targetting complex motif.2 Characterization of human and yeast Mak16 by EPR and Mössbauer spectroscopy revealed a redox-active [4Fe-4S]2+/1+ cluster with a midpoint potential below -450 mV. Yeast cells in which the endogenous Mak16 is substituted by its Fe/S-deficient C38A variant exhibited heightened sensitivity to redox stressors, including DTT, H₂O₂, and menadione. Our findings also reveal that upon binding to rRNA expansion segment 7a (ES7a) the redox properties of the nearby Fe/S cluster largely remain unchanged. However, disruption of Fe/S cluster coordination destabilized Mak16, impaired the Mak16-Rpf1 complex formation and decreased the 25S rRNA level. The critical role of Fe/S proteins in eukaryotic DNA replication and repair—through their involvement in DNA polymerases, primase, helicases, and glycosylase—has now been extended to ribosomal assembly. However, relying on such a vulnerable cofactor comes at a cost, as cluster loss can severely disrupt essential cellular processes. The inherent biosynthetic complexity and instability of Fe/S clusters allows a function as sensor for redox imbalance, creating new possibilities for Fe/S proteins to regulate cellular homeostasis.