The Fur protein is a primary regulator that monitors and controls cytoplasmic iron levels. We now report the identification of a regulatory pathway mediated by the Salmonella response regulator RstA that promotes Fur activity. Genome-wide expression experiments revealed that under iron-replete conditions, expression of the RstA protein from a plasmid lowered transcription levels of various genes involved in iron acquisition. The RstA protein controlled iron-responsive genes through the Fur-Fe(II) protein because deletion of the fur gene or iron depletion abrogated RstA-mediated repression of these genes. The RstA protein maintained wild-type levels of the Fur protein but exceptionally activated transcription of the feoAB operon encoding the ferrous iron transporter FeoB by binding directly to the feoA promoter. This FeoB induction resulted in increased ferrous iron uptake, which associates with the Fur protein because lack of RstA-dependent transcriptional activation of the feoA promoter and feoB-deletion abolished repression of the Fur target genes by the RstA protein. Under iron-replete conditions, RstA expression retarded Salmonella growth but enabled the Fur protein to repress the target genes beyond the levels which were simply accomplished by iron.