The safety and efficacy of a novel trivalent inactivated Salmonella vaccine consisting of Salmonella Enteritidis (SE), Salmonella Typhimurium (ST), and Salmonella Infantis (SI) was evaluated with chickens. Chickens were injected subcutaneously with 0.5 ml per dose of the vaccine with an oil adjuvant. To assess the efficacy of the vaccine, vaccinated and unvaccinated chickens were challenged by oral inoculation of SE, ST, SI, and Salmonella Heidelberg (SH) virulent strains 4 wk after vaccination. The vaccinated chickens showed no clinical abnormalities, and the body weight gain after vaccination was comparable to that of unvaccinated chickens. The shedding of SE from the vaccinated chickens at 4, 7, 10, and 14 days postchallenge were significantly reduced in comparison with those of the unvaccinated chickens. The efficacy of the vaccine against ST and SI was comparable to that against SE. The shedding of SH at 4, 7, 10, and 14 days postchallenge was also significantly reduced in the vaccinated chickens compared with those in the unvaccinated suggesting that the vaccine could be effective against different serotype strains having O antigens homologous to those of the vaccine strains. These results suggest that the novel trivalent inactivated vaccine can be an effective tool for controlling the Salmonella infections of O4, O7, and O9 groups in chicken farms.