We report here that the delivery of both alpha-galactosylceramide (alphaGalCer), 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 alphaGalCer-liposomes produce IL-10 when co-cultured with iNKT cells, whereas the cells treated with aqueous alphaGalCer 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 OVA323-339 peptide. Administration of alphaGalCer-OVA-liposomes into OVA-primed mice causes the development of CD4+ CD25(high) Foxp3+ T cells that produce both IL-10 and IFN-gamma, and induced the antigen-specific suppression of the secondary antibody responses when boosted with OVA alone. These results indicate that antigen-containing alphaGalCer-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.