The neonatal stage is characterized by weak responses to various infections and vaccines, thus the development of efficient formulas to improve vaccine effectiveness is of high priority. The glycolipid alpha galactosylceramide (αGalCer) is known as a potent immune modulator due mainly to natural killer (NK) T cell activation. Using a mouse tetanus toxoid (TT) immunization model, we observed that neonatal mice given αGalCer at the time of primary immunization on postnatal day (pnd) 17 had a significantly higher TT-specific immunoglobulin (Ig)M response as well as a memory IgG response, while αGalCer given on pnd 7 resulted in only marginal boosting. Consistently, immunostaining of the spleen sections from αGalCer-treated pnd 17 immunized neonates showed a higher number of Ki67+ cells in the splenic germinal centre area, suggesting a stronger response after immunization. In-vitro kinetic studies revealed that spleen cells from newborn to pnd 7 neonates did not respond to αGalCer stimulation, whereas cell proliferation was increased markedly by αGalCer after pnd 7, and became dramatic around neonatal pnd 17–18, which was accompanied by increased B, T and NK T cell populations in the spleen. In addition, in pnd 17 spleen cells, αGalCer significantly stimulated the production of NK T cytokines, interleukin (IL)-4 and interferon (IFN)-γ, and promoted the proliferation of CD23+ B cells, a subset of B cells enriched in germinal centres. These data suggest that αGalCer is an effective immune stimulus in the late neonatal stage, and thus may be useful in translational studies to test as a potential adjuvant to achieve a more efficient response to immunization.