Pathogenic Shigella spp. are the leading cause of bacterial dysentery, with Shigella flexneri and Shigella sonnei accounting for around 90% of cases worldwide. Although S. flexneri causes most disease in low-income countries (following ingestion of contaminated food and/or water), S. sonnei predominates in wealthy countries and is mainly spread from person to person. Although both species contain a large virulence plasmid, pINV, that is essential for the organism to cause disease, little is known about its maintenance. Here, using a counterselectable marker within the virulence-encoding region of pINV, we show that the S. sonnei plasmid is less stable than that of S. flexneri, especially at environmental temperatures. GmvAT, a toxin-antitoxin system, is responsible for the difference in stability and is present in pINV from S. flexneri but absent in S. sonnei pINV. GmvT is an acetyltransferase toxin that inhibits protein translation. Loss of GmvAT and a second toxin-antitoxin system, CcdAB, from pINV reduces S. sonnei plasmid stability outside the host, reflecting the host-adapted lifestyle and person-to-person transmission of this species and differences in its epidemiology.