Objective To investigate the regulatory effects of sodium butyrate on biofilm formation by Salmonella typhimurium and clarify its effects on the expression of virulence-related genes and pathogenicity under in vivo conditions.Methods S. typhimurium was treated with 4 mg/mL and 8 mg/mL sodium butyrate, and the effects of sodium butyrate on biofilm formation, motility, metabolic activity, and quorum sensing of the strain were detected. The expression changes of virulence genes (hilA, invF, and sipA) in sodium butyrate-treated strains were analyzed by quantitative real-time PCR. A C57BL/6 mouse infection model was established using S. typhimurium pretreated with 8 mg/mL sodium butyrate, and the survival time of mice, bacterial loads in the liver and spleen, and pathological changes in these organs were analyzed.Results Sodium butyrate at concentrations of 4 mg/mL and 8 mg/mL reduced biofilm formation by the strain in 96-well plates (P<0.05), decreased the diameters of bacterial colonies formed on semi-solid medium by 43.68% and 63.73%, respectively, reduced metabolic activity by 6.85% and 34.00%, respectively, and decreased purple pigment production by quorum sensing indicator bacteria by 42.15% and 67.46%, respectively. Treatment with 8 mg/mL sodium butyrate increased the expression levels of the hilA, invF, and sipA genes by 1.6-, 2.7-, and 1.3-fold, respectively, shortened the survival time of mice, increased bacterial loads in the liver and spleen, and caused obvious pathological changes in these organs.Conclusion Sodium butyrate can weaken biofilm formation by S. typhimurium through regulating its motility, quorum sensing, and metabolic activity, but it may also promote changes in the virulence of S. typhimurium.