BACKGROUND Salmonella Typhimurium, a non-host-adapted Gram-negative intracellular pathogen, is capable of infecting a variety of animal hosts and humans. OBJECTIVE This study utilized the prime-booster immunization strategy using Salmonella Typhimurium-LTB (S. Typhimurium-LTB) ghost with the aim of inducing a robust immune response for the prevention of avian salmonellosis. In addition, the effect of Montanide(TM) ISA 70VG adjuvant on S. Typhimurium-LTB ghost vaccination was investigated. ANIMALS AND METHODS A total of 75 chickens were divided into three groups (n=25) for intramuscular immunization: group A (non-immunized control injected with sterile PBS), group B (immunized with S. Typhimurium-LTB ghost), and group C (immunized with S. Typhimurium-LTB ghost plus Montanide(TM) ISA70VG adjuvant). RESULTS Compared with group A, the immunized chickens (groups B and C) exhibited increased titers of antigen specific plasma IgG and intestinal secretory IgA antibodies. In addition, group C showed enhanced induction of the humoral immune response compared to group B. The populations of splenic CD3+CD4+ and CD3+CD8+ T-cells increased significantly in both immunized groups. In addition, increased mRNA expression of the Th1 cytokines, IFN-γ, and IL-2 were observed in S. Typhimurium antigen-stimulated peripheral blood mononuclear cells from groups B and C chickens. Chickens from both vaccinated groups showed significant protection against virulent S. Typhimurium oral challenge compared to non-vaccinated chickens and a lower challenge strain count was recovered from the internal organs of group C. CONCLUSIONS Injection of S. Typhimurium-LTB ghost with or without Montanide(TM) ISA70VG adjuvant is capable of inducing protective immunity against the virulent S. Typhimurium infection in chickens.