Yersinia pestis has spilled over from wild rodent reservoirs to commensal rodents and humans, causing three historically recorded pandemics. Depletion in the copy number of the plasmid-encoded virulence gene pla occurred in later-dated strains of the first and second pandemics, yet the biological relevance of the pla deletion has been difficult to test. We identified modern Y. pestis strains that independently acquired the same pla depletion as ancient strains and herein show that excision of pla from the multicopy pPCP1 plasmid is accompanied by the integration of a separate full pPCP1 harboring pla into the single-copy pCD1 plasmid, reducing pla dosage. Moreover, we demonstrate that this depletion decreases the mortality of mice in models of bubonic plague but not in the pneumonic and septicemic forms of the disease. We hypothesize that pla depletion may have been selectively advantageous in bubonic plague, owing to rodent fragmentation after pandemic-induced mortality.