Although hemolytic lipids have been discovered from many human pathogens including Group B Streptococcus (GBS), strategies that neutralize their function are lacking. GBS is a leading cause of pregnancy-associated neonatal infections, and adult GBS infections are on the rise. The GBS hemolytic lipid toxin or granadaene, is cytotoxic to many immune cells including T and B cells. We previously showed that mice immunized with a synthetic nontoxic analog of granadaene known asR-P4had reduced bacterial dissemination during systemic infection. However, mechanisms important forR-P4mediated immune protection was not understood. Here, we show that immune serum fromR-P4-immunized mice facilitate GBS opsonophagocytic killing and protect naïve mice from GBS infection. Further, CD4+T cells isolated fromR-P4-immunized mice proliferated in response toR-P4stimulation in a CD1d- and iNKT cell-dependent manner. Consistent with these observations,R-P4immunized mice lacking CD1d or CD1d-restricted iNKT cells exhibit elevated bacterial burden. Additionally, adoptive transfer of iNKT cells fromR-P4vaccinated mice significantly reduced GBS dissemination compared to adjuvant controls. Finally, maternalR-P4vaccination provided protection against ascending GBS infection during pregnancy. These findings are relevant in the development of therapeutic strategies targeting lipid cytotoxins.