The PmrA-PmrB two-component system of Salmonella enterica controls resistance to the peptide antibiotic polymyxin B and to several antimicrobial proteins from human neutrophils. Transcription of PmrA-activated genes is induced by high iron, but can also be promoted by growth in low magnesium in a process that requires another two-component system, PhoP-PhoQ. Here, we define the genetic basis for the interaction between the PhoP-PhoQ and PmrA-PmrB systems. We have identified pmrD as a PhoP-activated gene that mediates the transcriptional activation of PmrA-regulated genes during growth in low magnesium. When transcription of pmrD is driven from a heterologous promoter, expression of PmrA-activated genes occurs even at repressing magnesium concentrations and becomes independent of the phoP and phoQ genes. The PmrD effect is specific for PmrA-regulated genes and requires functional PmrA and PmrB proteins. A pmrD mutant is sensitive to polymyxin if grown in low magnesium, but resistant if grown in high iron. The PmrD protein controls the activity of the PmrA-PmrB system at a post-transcriptional level.