The generation of multicellular behavior enhances the stress adaptability, antibiotic resistance, and pathogenic potential of Salmonella enterica serovar Typhimurium (S. Typhimurium), which is challenging for its prevention and control. Therefore, determination of the mechanism of multicellular behavior development is urgently required. Accordingly, this study investigated BolA, a transcription factor that promotes bacterial survival under different stresses. We found that BolA promoted the generation of multicellular behavior. Furthermore, transcriptome analysis revealed that BolA affected the expression of numerous genes, including biofilm formation and motility-related genes. In terms of biofilm formation, compared with the wild-type strain, bolA overexpression (269BolA+) increased the extracellular matrix content (extracellular polysaccharide, extracellular protein, and extracellular DNA (eDNA) by upregulating gene expression, ultimately increasing the biofilm formation ability by 2.56 times. For motility, bolA overexpression inhibited the expression of flagella synthesis genes, resulting in a 91.15 % decrease in motility compared with the wild-type (6 h). Further mechanistic analysis demonstrated that BolA affected the expression of the C-di-GMP pathway genes yeaJ and yhjH, which influenced the generation of multicellular behavior. In terms of biofilms, the extracellular polysaccharide content of 269BolA + ∆Yeaj (bolA overexpression and yeaJ deletion) was reduced by 89.91 % compared with 269BolA+, resulting in a 71.1 % reduction in biofilm forming ability. The motility of the 269∆BolA∆Yhjh (bolA/yhjH double deletion) strain was significantly decreased compared with that of 269∆BolA. Finally, the LacZ gene reporting showed that BolA promoted and inhibited the expression of yeaJ and yhjH, respectively. In conclusion, BolA mainly improves the content of extracellular polysaccharide by promoting the expression of yeaJ, thus enhancing the formation of biofilms. BolA also restricts flagellar synthesis by inhibiting yhjH expression, therefore reducing motility, ultimately promoting multicellular behavior arises. These findings lay a theoretical foundation for the prevention and control of S. Typhimurium.