Recombinant Saccharomyces cerevisiae strains expressing HIV antigens have shown promising pre-clinical results. Probiotic S. cerevisiae strains naturally induce gut immunity; thus, genetically engineered probiotic strains could be used to stimulate immune responses against HIV in the mucosa. Probiotic strains have a higher rate of heterologous protein production, meaning higher antigen's epitope expression levels per yeast cell. We expressed HIV-1 Gag protein in the probiotic yeasts' surface, which was eagerly phagocytosed by and induced type 1 polarization of human monocyte-derived dendritic cells (DCs) from healthy donors in vitro. We further matured DCs derived from HIV-1(+) donors with transformed yeasts and incubated them with autologous T cells. Only DCs matured with Gag-expressing probiotic strains were able to efficiently present antigen to CD8(+) T cells and induced their clonal expansion. Our results show that genetically engineered probiotic S. cerevisiae strains are a promising vaccination strategy against HIV.