Simple SummarySalmonella entericaserovar Enteritidis can cause human salmonellosis; however, its main carrier, poultry, does not show any clinical symptoms. In this study, we evaluated the potential of a mutantSalmonellaEnteritidis strain as a vaccine candidate. Compared to the wild-typeSalmonellaEnteritidis, the vaccine candidateSalmonellaEnteritidis strain was more attenuated. Moreover, single nucleotide polymorphism analysis revealed that genetic changes in the vaccine candidate strain corresponded with its phenotypes. Thus, theSalmonellaEnteritidis strain constructed in our laboratory might be valuable in developing a novel live-attenuated vaccine againstSalmonellaEnteritidis.AbstractSalmonellaEnteritidis is a common foodborne pathogen transmitted through poultry products, which are its main carriers. Poultry are vaccinated againstSalmonellaEnteritidis in many countries, despite the absence of clinical symptoms, using commercially available live-attenuated vaccines. We previously constructed a highly attenuated temperature-sensitive (ts)SalmonellaEnteritidis mutant, 2S-G10. In the present study, we describe the construction and attenuation-associated characteristics of 2S-G10. We infected 1-day-old chicks with 2S-G10 and the parental strains to evaluate the attenuation. One week after infection, 2S-G10 was not detected in the liver, cecum, or cecal tonsil tissues of the orally inoculated chicks, contrary to the parental strain. This indicates that 2S-G10 was highly attenuated when compared to the parental stain. In vitro experiments revealed the inability of 2S-G10 to grow at the normal body temperature of chickens and invade chicken liver epithelial cells. Moreover, single nucleotide polymorphism (SNP) analysis between the complete genome sequence of 2S-G10 and its parental strain revealed SNPs inbcsE,recG,rfaF, andpepD_1 genes, which are involved in epithelial cell invasion and persistence in host systems, growth, lipopolysaccharide core biosynthesis, and cellular survival under heat stress, respectively. These potential characteristics are consistent with the findings of in vitro experiments. Conclusively, chemical treatment–induced random genetic mutations highly attenuated 2S-G10, implying its potential to be developed as a novel live-attenuated vaccine againstSalmonellaEnteritidis.