Biogenesis of iron-sulfur (Fe-S) cluster-containing proteins relies on assistance of complex machineries. To date three systems, NIF, ISC, and SUF, were reported to allow maturation of Fe-S proteins. Here we report that the csdA-csdE (formally ygdK) genes of Escherichia coli constitute a sulfur-generating system referred to as CSD which also contributes to Fe-S biogenesis in vivo. This conclusion was reached by applying a thorough combination of both in vivo and in vitro strategies and techniques. Yeast two-hybrid analysis allowed us to show that CsdA and CsdE interact. Enzymology analysis showed that CsdA cysteine desulfurase activity is increased 2-fold in the presence of CsdE. Mass spectrometry analysis and site-directed mutagenesis showed that residue Cys-61 from CsdE acted as an acceptor site for sulfur provided by cysteine desulfurase activity of CsdA. Genetic investigations revealed that the csdA-csdE genes could act as multicopy suppressors of iscS mutation. Moreover, both in vitro and in vivo investigations pointed to a specific connection between the CSD system and quinolinate synthetase NadA.