ABSTRACT Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovars Typhimurium and Enteritidis, is responsible for a major global burden of invasive disease with high associated case-fatality rates. We recently reported the development of a candidate O-antigen–CRM 197 glycoconjugate vaccine against S. Typhimurium. Here, using a panel of mouse monoclonal antibodies generated by the vaccine, we examined the relative efficiency of different antibody isotypes specific for the O:4 antigen of S . Typhimurium to effect in vitro and in vivo killing of the invasive African S . Typhimurium strain D23580. All O:4-specific antibody isotypes could mediate cell-free killing and phagocytosis of S . Typhimurium by mouse blood cells. Opsonization of Salmonella with O:4-specific IgA, IgG1, IgG2a, and IgG2b, but not IgM, resulted in cell-dependent bacterial killing. At high concentrations, O:4-specific antibodies inhibited both cell-free complement-mediated and cell-dependent opsonophagocytic killing of S . Typhimurium in vitro . Using passive immunization in mice, the O:4-specific antibodies provided in vivo functional activity by decreasing the bacterial load in the blood and tissues, with IgG2a and IgG2b being the most effective isotypes. In conclusion, an O-antigen–CRM 197 glycoconjugate vaccine can induce O-antigen-specific antibodies of different isotypes that exert in vitro and in vivo killing of S . Typhimurium.