Innate cell-mediated immune responses are probably important to help prevent HIV-1 transmission to a new host across the genital mucosa. Invariant natural killer T (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid antigens presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, suggesting a role for iNKT cells in control of HIV-1. In this study we have investigated whether iNKT cells can directly detect HIV-infected dendritic cells (DCs) by using an HIV mutant lacking Nef and Vpu. Productive HIV-1 infection of DCs induced increased expression of CD1d as well as elevated intracellular levels of the endogenous glucocylceramide 24:1 iNKT cell antigen. This DC response occurred in a TLR-dependent manner and involved the modulation of enzymes in the sphingolipid pathway. iNKT cells were able to specifically detect and respond to rare productively HIV-1 infected DCs in a CD1d-dependent manner. This response was counteracted by Nef- and Vpu-mediated CD1d down-regulation. Furthermore, iNKT cells as well as DCs expressing both CD1d and HIV-receptors resided in the endometrial and cervical female genital mucosa, which are sites where HIV-1 transmission frequently occurs. Together, these findings indicate that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral antigen, and is actively targeted by viral immune evasion mechanisms.