Previously, we developed the monoclonal antibody (mAb) 449 after immunizing BALB/c mice with a live attenuated mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it helped protect against S. Typhimurium infection. Although the immunogen specific for mAb449 has been found to be localized in the lipopolysaccharide (LPS) region, it is not clear which part of LPS is involved in the defense against Salmonella infection. The present study aimed to identify sites recognized by mAb449. Electron microscopy analysis showed that mAb449 binds to the surface of Salmonella serotypes that express the O4 antigen, such as serovar Abortusequi and Paratyphi B. In addition, mAb449 enhanced the uptake of only O4-positive Salmonella serotypes within mouse macrophage-like cells. The results demonstrated that mAb449 is specific for the O4 antigen. We further verified the protective potential of mAb449 against O4-positive Salmonella serotypes in mice infection model, and found that mAb449 exhibited protective effect against the O4-positive Salmonella serotypes such as Abortusequi and Paratyphi B.