Neonates have a poorly developed immune system. Therefore it is important to develop vaccination strategies that induce protective immunity and immunological memory against pathogens early in life. The immunogenicity of a meningococcal serogroup C polysaccharide conjugate (MenC-CRM(197)) was assessed in neonatal mice, and effects of LT-K63 and CpG2006 and immunisation routes were compared. Neonatal mice were primed subcutaneously (s.c.) or intranasally (i.n.) with MenC-CRM(197) with or without LT-K63 or CpG2006 and re-immunised 16 and 30 days later by the same route and formulation. Antibody levels were measured and generation of immunological memory assessed by affinity maturation and kinetics of the Ab response. Serum bactericidal activity (SBA) was measured to evaluate protective efficacy. The second and third dose of MenC-CRM(197) mixed with either LT-K63 or CpG2006 induced a rapid increase in MenC-specific IgG antibodies, to levels higher than elicited by MenC-CRM(197) alone (P<0.01) and in unimmunised mice (P<0.001), indicating efficient generation of memory by priming through both s.c. and i.n. routes. SBA was detected after three s.c. immunisations with MenC-CRM(197) s.c. alone. However, only two doses of MenC-CRM(197)+LT-K63 or MenC-CRM(197)+CpG2006 were needed to induce SBA levels>16. LT-K63 and CpG2006 enhanced neonatal antibody responses, affinity maturation, immunological memory to the conjugate MenC-CRM(197) and protective immunity. These results encourage the development of neonatal vaccination strategies to induce protective immunity and immunological memory against meningococcal disease.