Despite the prominence of iron-sulfur cluster (ISC) proteins in bioenergetics, intermediary metabolism, and redox regulation of cellular, mitochondrial, and nuclear processes, these proteins have been given scarce attention in Drosophila. Moreover, biosynthesis and delivery of ISCs to target proteins requires a highly regulated molecular network that spans different cellular compartments. The only Drosophila ISC biosynthetic protein studied to date is frataxin, in attempts to model Friedreich's ataxia, a disease arising from reduced expression of the human frataxin homologue. One of several proteins involved in ISC biogenesis is heat shock protein cognate 20 (Hsc20). Here we characterize two piggyBac insertion mutants in Drosophila Hsc20 that display larval growth arrest and deficiencies in aconitase and succinate dehydrogenase activities, but not in isocitrate dehydrogenase activity; phenotypes also observed with ubiquitous frataxin RNA interference. Furthermore, a disruption of iron homeostasis in the mutant flies was evidenced by an apparent reduction in induction of intestinal ferritin with ferric iron accumulating in a subcellular pattern reminiscent of mitochondria. These phenotypes were specific to intestinal cell types that regulate ferritin expression, but were notably absent in the iron cells where ferritin is constitutively expressed and apparently translated independently of iron regulatory protein 1A. Hsc20 mutant flies represent an independent tool to disrupt ISC biogenesis in vivo without using the RNA interference machinery.