HIV-1 has evolved several strategies to subvert host immune responses to the infected cells. One is to inhibit CTL recognition by HIV-1 Nef-mediated down-regulation of MHC-I expression on the surface of infected cells. Here we report that Nef also reduces the expression of the non-classical MHC-I like CD1d molecule, a third lineage of antigen-presenting molecule, which presents lipid antigens. Nef achieves this by increasing internalization of CD1d molecules from the cell surface and retaining CD1d in the trans-Golgi-network (TGN). This effect depends on a tyrosine-based motif present in CD1 cytoplasmic tail as well as the actions of four Nef motifs, which are known to be involved in the down-regulation of MHC-I or CD4. These results suggest that Nef regulates intracellular trafficking of CD1d via a distinct but shared pathway with MHC-I and CD4. Thus, HIV-1 reduces the visibility of its infected cells not only to MHC-I-restricted T cells but also to CD1d-restricted NKT cells. Given that CD1d-restricted T cells have unique effector and regulatory functions in innate and adapted immune responses as compared with their counterpart MHC-restricted T cells, our data provide additional new insights into molecular basis of HIV-1-mediated damage to the immune system.