The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway functions to incorporate inorganic Fe-S cofactors into a variety of proteins, including several DNA repair enzymes. However, the mechanisms regulating the CIA pathway are unknown. We describe here that the MAGE-F1-NSE1 E3 ubiquitin ligase regulates the CIA pathway through ubiquitination and degradation of the CIA-targeting protein MMS19. Overexpression or knockout of MAGE-F1 altered Fe-S incorporation into MMS19-dependent DNA repair enzymes, DNA repair capacity, sensitivity to DNA-damaging agents, and iron homeostasis. Intriguingly, MAGE-F1 has undergone adaptive pseudogenization in select mammalian lineages. In contrast, MAGE-F1 is highly amplified in multiple human cancer types and amplified tumors have increased mutational burden. Thus, flux through the CIA pathway can be regulated by degradation of the substrate-specifying MMS19 protein and its downregulation is a common feature in cancer and is evolutionarily controlled.