One important event for the divergence of Salmonella from Escherichia coli was the acquisition by horizontal transfer of the Salmonella pathogenicity island 1 (SPI-1), containing genes required for the invasion of host cells by Salmonella. HilD is an AraC-like transcriptional regulator in SPI-1 that induces the expression of the SPI-1 and many other acquired virulence genes located in other genomic regions of Salmonella. Additionally, HilD has been shown to positively control the expression of some ancestral genes (also present in E. coli and other bacteria), including phoH. In this study, we determined that both the gain of HilD and cis-regulatory evolution led to the integration of the phoH gene into the HilD regulon. Our results indicate that a HilD-binding sequence was generated in the regulatory region of the S. enterica serovar Typhimurium phoH gene, which mediates the activation of promoter 1 of this gene under SPI-1-inducing conditions. Furthermore, we found that repression by H-NS, a histone-like protein, was also adapted on the S. Typhimurium phoH gene and that HilD activates the expression of this gene in part by antagonizing H-NS. Additionally, our results revealed that the expression of the S. Typhmurium phoH gene is also activated in response to low phosphate but independently of the PhoB/R two-component system, known to regulate the E. coli phoH gene in response to low phosphate. Thus, our results indicate that cis-regulatory evolution has played a role in the expansion of the HilD regulon and illustrate the phenomenon of differential regulation of ortholog genes. IMPORTANCE Two mechanisms mediating differentiation of bacteria are well known: acquisition of genes by horizontal transfer events and mutations in coding DNA sequences. In this study, we found that the phoH ancestral gene is differentially regulated between Salmonella Typhimurium and Escherichia coli, two closely related bacterial species. Our results indicate that this differential regulation was generated by mutations in the regulatory sequence of the S. Typhimurium phoH gene and by the acquisition by S. Typhimurium of foreign DNA encoding the transcriptional regulator HilD. Thus, our results, together with those from an increasing number of studies, indicate that cis-regulatory evolution can lead to the rewiring and reprogramming of transcriptional regulation, which also plays an important role in the divergence of bacteria through time.