Salmonella typhimurium is the causative agent of bacterial gastroenteritis in human via livestock products. S. typhimurium mutants (STD4 and STD5) deleted the hmpA, rpoS, ssrAB, aroA and ppk genes were constructed and, attenuated virulence and protective efficacy of the multiple mutants were evaluated in mice as a novel vaccine candidate. In the virulence test, CFUs of STD4 (hmpA, rpoS, ssrAB, and aroA) and STD5 recovered from the liver and spleen of the mice were highly decreased at the range of 2.40 to 5.51 logs for 15 days postinfection.  The changes of organ/body weight ratios in mice were also showed same trends as the changes of CFUs in virulence test. In the protection test, CFUs of STD4 and STD5 recovered from the organs in mice were highly decreased compared to non-vaccination group. The Polyphospate kinase (ppk) gene was not associated with protective efficacy but attenuated virulence in S. typhimurium mutant. Our results indicated that STD4 and STD5 firstly developed in this study showed highly attenuated virulence and protective efficacy. This work shows that S. typhimurium mutant deleted the multiple genes provide a basis for further development of a novel vaccine.