Select nontyphoidal Salmonella enterica (NTS) serotypes were recently found to encode the Salmonella cytolethal distending toxin (S-CDT), an important virulence factor for serotype Typhi, the causative agent of typhoid fever. Using a PCR-based assay, we determined that among 21 NTS serotypes causing the majority of food-borne salmonellosis cases in the United States, genes encoding S-CDT are conserved in isolates representing serotypes Javiana, Montevideo, and Oranienburg but that among serotype Mississippi isolates, the presence of S-CDT-encoding genes is clade associated. HeLa cells infected with representative strains of these S-CDT-positive serotypes had a significantly higher proportion of cells arrested in the G2/M phase than HeLa cells infected with representative strains of S-CDT-negative serotypes Typhimurium, Newport, and Enteritidis. The G2/M cell cycle arrest was dependent on CdtB, the active subunit of S-CDT, as infection with isogenic ΔcdtB mutants abolished their ability to induce a G2/M cell cycle arrest. Infection with S-CDT-encoding serotypes was significantly associated with activation of the host cell’s DNA damage response (DDR), a signaling cascade that is important for detecting and repairing damaged DNA. HeLa cell populations infected with S-CDT-positive serotypes had a significantly higher proportion of cells with DDR protein 53BP1 and γH2AX foci than cells infected with either S-CDT-negative serotypes or isogenic ΔcdtB strains. Intoxication with S-CDT occurred via autocrine and paracrine pathways, as uninfected HeLa cells among populations of infected cells also had an activated DDR. Overall, we show that S-CDT plays a significant role in the cellular outcome of infection with NTS serotypes.