DNA phosphorothioate (PT) modification, in which the non-bridging oxygen of the sugar-phosphate backbone is substituted by sulfur, occurs naturally in diverse bacteria and archaea and is regulated by the DndABCDE proteins. DndABCDE and the restriction cognate DndFGHI constitute a new type of defense system that prevents the invasion of foreign DNA in Salmonella enterica serovar Cerro 87. GAAC/GTTC consensus contexts across genomes were found to possess partial PT modifications even in the presence of restriction activity, indicating the regulation of PT. The abundance of PT in cells must be controlled to suit cellular activities. However, the regulatory mechanism of PT modification has not been characterized. The result here indicated that genomic PT modification in S. enterica is controlled by the transcriptional regulator DndB, which binds to two regions in the promoter, each possessing a 5'-TACGN(10)CGTA-3' palindromic motif, to regulate the transcription of dndCDE and its own gene. Site-directed mutagenesis showed that the Cys29 residue of DndB plays a key role in its DNA-binding activity or conformation. Proteomic analysis identified changes to a number of cellular proteins upon up-regulation and loss of PT. Considering the genetic conservation of dnd operons, regulation of PT by DndB might be widespread in diverse organisms.