While human cells express potent antiviral proteins as part of the host defense repertoire, viruses have evolved their own arsenal of proteins to antagonize them. BST2 was identified as an inhibitory cellular protein of HIV-1 replication, which tethers virions to the cell surface to prevent their release. On the other hand, the HIV-1 accessory protein, Vpu, has the ability to downregulate and counteract BST2. Vpu also possesses the ability to downmodulate cellular CD4 and SLAMF6 molecules expressed on infected cells. However, the role of Vpu in HIV-1 infection in vivo remains unclear. Here, using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrate that Vpu contributes to the efficient spread of HIV-1 in vivo during the acute phase of infection. Although Vpu did not affect viral cytopathicity, target cell preference, and the level of viral protein expression, the amount of cell-free virions in vpu-deficient HIV-1-infected mice was profoundly lower than that in wild-type HIV-1-infected mice. We provide a novel insight suggesting that Vpu concomitantly downregulates BST2 and CD4, but not SLAMF6, from the surface of infected cells. Furthermore, we show evidence suggesting that BST2 and CD4 impair the production of cell-free infectious virions but do not associate with the efficiency of cell-to-cell HIV-1 transmission. Taken together, our findings suggest that Vpu downmodulates BST2 and CD4 in infected cells and augments the initial burst of HIV-1 replication in vivo. This is the first report demonstrating the role of Vpu in HIV-1 infection in an in vivo model.