ABSTRACT Nontyphoidal salmonellae, particularly Salmonella enterica serovar Typhimurium, are a major cause of invasive disease in Africa, affecting mainly young children and HIV-infected individuals. Glycoconjugate vaccines provide a safe and reliable strategy against invasive polysaccharide-encapsulated pathogens, and lipopolysaccharide (LPS) is a target of protective immune responses. With the aim of designing an effective vaccine against S . Typhimurium, we have synthesized different glycoconjugates, by linking O-antigen and core sugars (OAg) of LPS to the nontoxic mutant of diphtheria toxin (CRM ₁₉₇ ). The OAg-CRM ₁₉₇ conjugates varied in (i) OAg source, with three S . Typhimurium strains used for OAg extraction, producing OAg with differences in structural specificities, (ii) OAg chain length, and (iii) OAg/CRM ₁₉₇ ratio. All glycoconjugates were compared for immunogenicity and ability to induce serum bactericidal activity in mice. In vivo enhancement of bacterial clearance was assessed for a selected S . Typhimurium glycoconjugate by challenge with live Salmonella . We found that the largest anti-OAg antibody responses were elicited by (i) vaccines synthesized from OAg with the highest glucosylation levels, (ii) OAg composed of mixed- or medium-molecular-weight populations, and (iii) a lower OAg/CRM ₁₉₇ ratio. In addition, we found that bactericidal activity can be influenced by S . Typhimurium OAg strain, most likely as a result of differences in OAg O-acetylation and glucosylation. Finally, we confirmed that mice immunized with the selected OAg-conjugate were protected against S . Typhimurium colonization of the spleen and liver. In conclusion, our findings indicate that differences in the design of OAg-based glycoconjugate vaccines against invasive African S . Typhimurium can have profound effects on immunogenicity and therefore optimal vaccine design requires careful consideration.