AbstractDendritic cells (DCs) play an important role in viral infections both as initiators of immunity and as viral targets. Interaction between DCs and the innate-like CD1d-restricted natural killer T (NKT) cells results in the mutual activation of both cells and the subsequent initiation of cellular immune responses. Here, we show that HIV-1 inhibits the surface expression of CD1d in productively infected DCs and identify this as a novel activity of the HIV-1 vpu gene product. Interestingly, the viral protein U (Vpu) does not enhance constitutive CD1d endocytosis or induce rapid CD1d degradation. Instead, the Vpu protein interacts with CD1d and suppresses its recycling from endosomal compartments to the cell surface by retaining CD1d in early endosomes. This interference with the CD1d antigen presentation pathway strongly inhibits the ability of infected DCs to activate CD1d-restricted NKT cells. Given that the interaction with CD1d-expressing DCs is central to the ability of NKT cells to regulate immunity, these data suggest that interference with the CD1d antigen presentation pathway represents an HIV-1 strategy to evade innate cellular immune responses and imply a role for the innate-like CD1d-restricted NKT cells in the host defense against HIV-1.