[Background] Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic pathogen that causes a variety of food-borne diseases. The biological function of ybiH gene in S. typhimurium has not been determined. [Objective] In order to define the pathogenicity of ybiH gene to S. typhimurium，the ybiH gene mutant and complementary strain of S. typhimurium were constructed and characterized. [Methods] In this paper, the mutant STΔybiH of S. typhimurium CVCC541 standard strain was successful constructed by λ-Red homologous recombination system. At the same time, its complementary strain STΔybiH/pybiH was successfully constructed. Then we analyzed the biological characteristics of growth characteristics, motility, biochemical properties and virulence. [Results] The results showed that the growth rate of STΔybiH was slightly faster than that of the standard strain and the complementary strain, but there was no significant difference in motility and biochemical characteristics. However, the deletion of ybiH gene significantly increased the adhesion and invasion of S. typhimurium to IEC-6 cells and RAW 264.7 cells. The results of qRT-PCR showed that the expression of InvH gene was significantly increased in the deletion strain. The results showed that the deletion of ybiH gene increased the invasiveness of S. typhimurium. In addition, the intracellular growth rate of the standard strain and the deletion strain did not change significantly in the intracellular survival experiment, indicating that the deletion of the ybiH gene had little effect on the survival of Salmonella in RAW 264.7 cells. [Conclusion] The ybiH gene mediates the adhesion and invasiveness of S. typhimurium, which provides a theoretical basis for further elucidating the function of ybiH gene.