Protective efficacy of trivalent Salmonella inactivated vaccine containing Chlorhexidine-inactivated S. Enterltidis (SE), S. Typhimurium (ST), and S. Gallinarum (SG) strains, was evaluated in this study. A total of 70 brown nick layers were divided into 7 groups, A to G, containing 10 hens per group. All hens in groups B to D were intramuscularly immunized with approximately 7×10 8 cells (3×10 8 cells of SE+1×10 8 cells of ST+3×10 8 cells of SG) of the trivalent vaccine in 0.5 mL of PBS. All chickens in groups E to G were injected with sterile PBS. All hens of groups B and E, groups C and F, and groups D and G were orally challenged with approximately 2 ×10 9 CFU of wild-type SE, ST, and SG, respectively. Serum IgG titers and CD3 + CD4 + T-cells, and CD3 + CD8 + T-cells levels of groups B to D significantly higher than those of group A. In addition, all animals in groups A to C, E and F showed no clinical symptoms and survived after the virulent challenges, whereas one chicken in group D died and all chickens in group G died following the challenge. The protection against wild-type SE and ST in liver, spleen, cecum, and cloaca of groups B and C chickens was significant effective as compared with those in groups E and F. These indicate that the trivalent inactivated vaccine can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and cellular immune response in chickens.