Abstract The PhoPQ two-component regulatory system coordinates the response of Salmonella enterica serovar Typhimurium to diverse environmental challenges encountered during infection of hosts, including changes in Mg2+ concentrations, pH, and antimicrobial peptides. Moreover, PhoPQ-dependent regulation of gene expression promotes intracellular survival of Salmonella in macrophages, and contributes to the resistance of this pathogen to reactive nitrogen species (RNS) generated from the nitric oxide produced by the inducible nitric oxide (NO) synthase of macrophages. We report here that Salmonella strains with mutations of phoPQ are hypersensitive to killing by RNS generated in vitro. The increased susceptibility of ∆phoQ Salmonella to RNS requires molecular O2 and coincides with the nitrotyrosine formation, the oxidation of [4Fe-4S] clusters of dehydratases, and DNA damage. Mutations of respiratory NADH dehydrogenases prevent nitrotyrosine formation and abrogate the cytotoxicity of RNS against ∆phoQ Salmonella, presumably by limiting the formation of peroxynitrite (ONOO−) arising from the diffusion-limited reaction of exogenous NO and endogenous superoxide (O2 •−) produced in the electron transport chain. The mechanism underlying PhoPQ-mediated resistance to RNS is linked to the coordination of Mg2+ homeostasis through the PhoPQ-regulated MgtA transporter. Collectively, our investigations are consistent with a model in which PhoPQ-dependent Mg2+ homeostasis protects Salmonella against nitrooxidative stress.