Mouse natural killer T cells with an invariant Valpha14-Jalpha18 TCR rearrangement (Valpha14i NKT cells) are able to regulate immune responses through rapid and large amounts of Th1 and Th2 cytokine production. It has been reported that in vivo administration of the Valpha14i NKT cell ligand, alpha-galactosylceramide (alpha-GalCer) significantly reduced morbidity and mortality of acute graft-versus-host disease (GVHD) in mice. In this study, we examined whether adoptive transfer of in vitro-expanded Valpha14i NKT cells using alpha-GalCer and IL-2 could modulate acute GVHD in the transplantation of spleen cells of C57BL/6 mice into (B6xDBA/2) F(1) mice. We found that the adoptive transfer of cultured spleen cells with a combination of alpha-GalCer and IL-2, which contained many Valpha14i NKT cells, modulated acute GVHD by exhibiting long-term mixed chimerism and reducing liver damage. Subsequently, the transfer of Valpha14i NKT cells purified from spleen cells cultured with alpha-GalCer and IL-2 also inhibited acute GVHD. This inhibition of acute GVHD by Valpha14i NKT cells was blocked by anti-IL-4 but not by anti-IFN-gamma monoclonal antibody. Therefore, the inhibition was dependent on IL-4 production by Valpha14i NKT cells. Our findings highlight the therapeutic potential of in vitro-expanded Valpha14i NKT cells for the prevention of acute GVHD after allogeneic hematopoietic stem cell transplantation.