Commercial bivalent killed Salmonella vaccine Salenvac-T has been used in several countries in order to prevent salmonellosis with Salmonella enterica serovars Enteritidis (SE) and Typhimurium (ST) in poultry. However, this vaccine has not been used in poultry farms in South Korea. In this study, we evaluated the efficacy of Salenvac- T vaccine to protect against the challenge of virulent SE and ST, and the effect of the vaccine on egg production and mortality in layer hens. The colonization of liver, spleen and cecum with challenged SE and ST was reduced in vaccinated chickens compared with that of unvaccinated control group. The twice vaccination with Salenvac-T induced elevated antibody responses against both SE and ST detected by enzyme-linked immunosorbent assay (ELISA). The higher average hen-day production was observed in the vaccinated layer hens than in the unvaccinated layer hens without significance. The average mortality was lower in the vaccinated layer hens during the experiment period. The antibody responses to both SE and ST were persistently detected in the vaccinated layers. In summary, vaccination with Salenvac-T reduces colonization of internal organs and induces good antibody responses, thereby results in higher performance and lower egg contamination with SE and ST in layer hens.