Abstract Salmonella spp. infections transmitted by contaminated poultry and eggs represent a major global health burden. Salmonella enterica serovar. Enteritidis is the leading cause of human salmonellosis worldwide. The cell surface antigens of Salmonella Enteritidis play an important role in the host–pathogen interactions and as such represent potential candidates for subunit-vaccine development. An immunogenic subcellular extract obtained from whole Salmonella Enteritidis cells (HE) was encapsulated in nanoparticles made with the polymer Gantrez (HE-NP). Proteomics was used to investigate the complex protein nature of the HE extract. Immunogenicity and protection studies against lethal Salmonella Enteritidis challenge were performed in BALB/c mice. Increased survival was observed in vaccinated mice as compared to a control group; 80% of the mice immunized with the HE-NP formulation survived even when administered 49 days before the lethal challenge. The cytokines released from in vitro-stimulated spleens showed a strong gamma interferon response in all immunized groups at day 10 post-immunization. However, the immunity induced by HE-NP at day 49 post-immunization suggests the involvement of a TH2 subclass in the protective effect. The potential for mucosal vaccination suggests that HE-nanoparticles may represent an important alternative to the conventional attenuated vaccines against Salmonella Enteritidis.