Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid, a notifiable infectious disease in poultry. However, the pathogenic mechanism of SG-induced systemic infection in chickens remains unclear. Thioredoxin reductase (TrxB) is a redox protein crucial for regulating various enzyme activities in Salmonella serovar, but the role in SG-induced chicken systemic infection has yet to be determined. Here, we constructed a mutant SG strain lacking the trxB gene (trxB::Cm) and used chicken embryo inoculation and chicken oral infection to investigate the role of trxB gene in the pathogenicity of SG. Our results showed that trxB::Cm exhibited no apparent differences in colony morphology and growth conditions but exhibited reduced tolerance to H₂O₂ and increased resistance to bile acids. In the chicken embryo inoculation model, there was no significant difference in the pathogenicity of trxB::Cm and wild-type (WT) strains. In the chicken oral infection, the WT-infected group exhibited typical clinical symptoms of fowl typhoid, with complete mortality between days 6 and 9 post infection. In contrast, the trxB::Cm group showed a 100% survival rate, with no apparent clinical symptoms or pathological changes observed. The viable bacterial counts in the liver and spleen of the trxB::Cm-infected group were significantly reduced, accompanied by decreased expression of cytokines and chemokines (IL-1β, IL-6, IL-12, CXCLi1, TNF-α, and IFN-γ), which were significantly lower than those in the WT group. These results show that the pathogenicity of the trxB-deficient strain was significantly attenuated, indicating that the trxB gene is a crucial virulence factor in SG-induced systemic infection in chickens, suggesting that trxB may become a potentially effective target for controlling and preventing SG infection in chickens.