KRN7000, or α-GalCer, is a potent agonist for natural killer T (NKT) cells. The 3-hydroxyl group of its phytosphingosine moiety is important for activating NKT cells, whereas its 4-hydroxyl group is perceived to be less crucial. To experimentally determine the role of the 4-hydroxyl group, we synthesized the 3-deoxy analogue of α-GalCer. It was found that 3-deoxy-α-GalCer induced potent cytokine responses from NKT cells, comparable to those of both α-GalCer and 4-deoxy-α-GalCer. This result and our docking studies suggest that the effects of an absence of the 3-hydroxyl group are compensated by the presence of a hydroxyl group at the C-4 position. Thus, we conclude that the 4-hydroxyl group of α-GalCer is as important to the mechanism of action as the 3-hydroxyl group and that the two hydroxyl groups could play individual and cooperative roles in orienting the glycolipid into the proper position in CD1d to be recognized by the T cell receptor of NKT cells.