Three precisely defined deletion mutants of Salmonella enterica serovar Enteritidis were constructed, a guanine auxotrophic DeltaguaB mutant, a nonflagellated DeltafliC mutant, and an auxotrophic and nonflagellated DeltaguaB DeltafliC double mutant. All three mutants were less invasive than the wild-type strain in primary chicken cecal epithelial cells and the human epithelial cell line T84 and less efficiently internalized in the chicken macrophage cell line HD11. The DeltafliC mutant was pathogenic in orally infected BALB/c mice, while the DeltaguaB mutant was attenuated and conferred protection against a challenge with the pathogenic parent strain. The DeltaguaB DeltafliC double mutant was totally asymptomatic and conferred better protection than the DeltaguaB mutant. This indicates that the major flagellar protein flagellin is not required for efficient vaccination of BALB/c mice against Salmonella infection. The DeltaguaB DeltafliC mutant was also safe for vaccination of 1-day-old chickens. After two immunizations, it induced statistically significant protection against infection of the internal organs of the birds by a virulent S. enterica serovar Enteritidis challenge strain but not against intestinal colonization. These data demonstrate that nonflagellated attenuated Salmonella mutants can be used as marker vaccines.