In this report, we demonstrate that H-NS is essential for establishing the Mg(2+)-responsive transcriptional regulation of the PhoP regulon in Salmonella. Deletion of this regulatory gene abolished the transcriptional repression of PhoP-activated genes when bacteria were grown in high environmental Mg(2+), thus stimulating expression of phoP and other PhoP regulon genes. In the absence of H-NS, transcriptional activation was PhoP-dependent for those genes only activated by PhoP, but was PhoP-independent for those genes activated by both PhoP and SlyA. The H-NS protein footprints the phoP promoter in a sequence located upstream of the PhoP box; mutation of this cis-acting factor abolished transcriptional repression of the phoP gene equivalent to the phenotype exhibited in the hns mutant. Further results showed that H-NS gel shifts other PhoP regulon promoters, indicating that a PhoP-activated gene would be transcriptionally repressed via direct H-NS binding and inhibition of its activator PhoP. Furthermore, H-NS footprints a newly identified SlyA box and the reverse PhoP box in the pagC promoter, suggesting that both SlyA and PhoP compete with this regulatory protein. Therefore, H-NS should pair with SlyA and PhoP to establish a forward regulatory loop to regulate expression of pagC, and perhaps other PhoP- and SlyA-dependent genes.