We report here that the delivery of both α-galactosylceramide (αGalCer), a representative ligand for invariant natural killer T (iNKT) cells, and an antigenic polypeptide to marginal zone B cells induces the differentiation of regulatory cells in vivo, and suppresses the secondary antibody responses in mice. Splenic CD21⁺ CD23^- B cells of mice treated with αGalCer-liposomes produce IL-10 when co-cultured with iNKT cells, whereas the cells treated with aqueous αGalCer fail to do so. Adoptive transfer of the B cells into syngenic mice leads to the expansion of splenic CD11c^low CD45RB^high cells, which convert naive CD4⁺ T cells from RAG2-deficient DO11.10 mice to CD4⁺ CD25^high Foxp3⁺ T cells in the presence of OVA_323-339 peptide. Administration of αGalCer-OVA-liposomes into OVA-primed mice causes the development of CD4⁺ CD25^high Foxp3⁺ T cells that produce both IL-10 and IFN-γ, and induced the antigen-specific suppression of the secondary antibody responses when boosted with OVA alone. These results indicate that antigen-containing αGalCer-liposomes can facilitate the development of tolerogenic antigen-presenting cells and inducible regulatory T cells that are involved in the suppression of immune responses to antigens.