Salmonellosis remains a major medical and an unmet socioeconomic challenge. Worldwide, more than three million deaths per year are associated with Salmonella enterica serovar Enteritidis infections. Although commercially available vaccines for use in poultry exist, their efficacy is limited. We previously described a method for isolating a heat extract (HE) fraction of the cell surface of S. Enteritidis that contained major antigenic complexes immunogenic in hens naturally infected with the bacterium. One single dose of S. Enteritidis' HE induced protection against lethal salmonellosis in mice. Furthermore, HE encapsulation in nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (PVM/MA), Gantrez AN, improved and prolonged the protection against the disease in mice. We formulated new preparations of Gantrez AN nanoparticles with HE S. Enteritidis and assessed their stability in drinking water and their efficacy in hens after experimental infection. The oral treatment of six-week-old hens with two doses of HE nanoparticles significantly reduced the Salmonella excretion in hens. Due to the effectiveness of the treatment in reducing bacterial excretion, we conclude that HE nanoencapsulation obtained from S. Enteritidis is a viable novel vaccination approach against salmonellosis in farms.