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.