Salmonella Enteritidis (SE) has been a major causative agent of food-borne human disease due to consumption of contaminated eggs and poultry meat. To prevent SE infection in poultry, and therefore minimize human infections, vaccination with either killed or live SE vaccine is suggested. We evaluated a newly developed killed bacterin using a representative SE isolate in Korea. Among pool of SE isolates, two highly virulent isolates (the one isolate from chicken, the other from human) were selected by measuring mortality in mouse and chickens administered. The chickens were injected intramuscularly with killed vaccine and were challenged with highly virulent SE strain 3 week after vaccination. The recovered colony count (cfu/g) of spleen and cecal content in the vaccinated groups was reduced compared with those of the unvaccinated control group. The antibody level in the vaccinated groups was higher at 3 week post vaccination. These results indicate that vaccination with killed vaccine was effective in preventing the infection of virulent SE. Further study for a large number of layers should be needed for the effect of egg production, SE shedding in feces, persistence of antibody level.