Yersinia pestis, the causative agent of plague, evolved from the closely related pathogen Yersinia pseudotuberculosis During its emergence, Y. pestis is believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system. Y. pseudotuberculosis and the basal human-avirulent strains of Y. pestis harbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation of Y. pestis to stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability of Y. pestis to survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele of phoP (phoP-S215), a derivative carrying the basal allele (phoP-G215) exhibited slightly defective growth under a low-Mg2+ condition and decreased transcription of a PhoP target gene, ugd, as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. The phoP-G215 strain showed no apparent defect in flea colonization, although a phoP-null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence in Y. pestisIMPORTANCEY. pestis acquired a single nucleotide polymorphism (SNP) in phoP when the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show that Y. pestis carrying the modern phoP allele has an increased ability to induce the PhoP-regulated ugd gene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence in Y. pestis Additionally, we present the first evidence that phoP confers a survival fitness advantage to Y. pestis inside the flea midgut.