[Background] Salmonella typhimurium is an important zoonotic pathogen that causes economically devastating to livestock breeding and potential threats to human health. Regulatory proteins are involved in the regulation of the survival and infection of pathogens. [Objective] To analyze the effect of regulatory protein RstB on the biological characteristics and pathogenicity, the rtsB gene mutant and complemented strain of S. typhimurium were constructed and characterized. [Methods] We constructed the rtsB gene mutant strain and complemented strain of S. typhimurium SAT52 using the Red recombination system and complementary plasmid. Then we analyzed the growth characteristics, motility, biofilm formation, cells adhesion and invasion ability, and pathogenicity. [Results] The deletion of rtsB gene did not affect the growth rate of SAT52. However, the mutant strain showed significantly enhanced motility and decreased biofilm formation ability. Compared with the wild-type strain, the capacities of adherent to Hela cells and survival in RAW264.7 cells were significantly decreased for the mutant strain. The results of animal infection experiments showed that the virulence of mutant strain was attenuated. [Conclusion] The rtsB gene plays an important role in the pathogenesis of S. typhimurium infection, providing a certain basis for further understanding of the pathogenic mechanism of S. typhimurium. These data indicated that regulator RtsB plays an important role in the process of S. typhimurium infection, which would help us to comprehensive understand the pathogenic mechanism of S. typhimurium.