SAMHD1 is a myeloid-specific protein that restricts HIV-1 infection by inhibiting an early step of the viral life cycle. It is counteracted by the primate lentivirus accessory protein Vpx, which induces proteasomal degradation of SAMHD1. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells, and silencing of SAMHD1 increases the susceptibility of monocytic-derived dendritic cells to HIV-1 infection. SAMHD1 is highly expressed in HIV-1 non-permissive cells such as THP-1, monocytes, and monocyte-derived dendritic cells (MDDCs), but is absent from HIV-1-sensitive T-cell lines. SAMHD1 is also expressed in monocyte-derived macrophages (MDMs), but at lower levels than in their monocyte precursor. Vpx from SIVSM and HIV-2 lineages can degrade SAMHD1, while Vpx from SIVRCM of red-capped mangabeys cannot. SAMHD1 silencing in THP-1 cells increases their permissiveness to HIV-1 infection, and SAMHD1 expression in U937 cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is likely required for HIV-1 restriction. SAMHD1 restricts HIV-1 infection in primary MDDCs, and its silencing increases the susceptibility of these cells to HIV-1. SAMHD1 is involved in nucleotide metabolism and may degrade or prevent accumulation of HIV DNA. SAMHD1 and TREX1 are both involved in the cell-intrinsic antiviral response and are associated with Aicardi-Goutières syndrome. SAMHD1 is a key factor in the fate of HIV-1 infection in myeloid cells, and modulating its function could enhance innate and adaptive immune responses. These findings suggest that targeting SAMHD1 could be a strategy for developing dendritic-cell-targeted vaccines against HIV/AIDS.SAMHD1 is a myeloid-specific protein that restricts HIV-1 infection by inhibiting an early step of the viral life cycle. It is counteracted by the primate lentivirus accessory protein Vpx, which induces proteasomal degradation of SAMHD1. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells, and silencing of SAMHD1 increases the susceptibility of monocytic-derived dendritic cells to HIV-1 infection. SAMHD1 is highly expressed in HIV-1 non-permissive cells such as THP-1, monocytes, and monocyte-derived dendritic cells (MDDCs), but is absent from HIV-1-sensitive T-cell lines. SAMHD1 is also expressed in monocyte-derived macrophages (MDMs), but at lower levels than in their monocyte precursor. Vpx from SIVSM and HIV-2 lineages can degrade SAMHD1, while Vpx from SIVRCM of red-capped mangabeys cannot. SAMHD1 silencing in THP-1 cells increases their permissiveness to HIV-1 infection, and SAMHD1 expression in U937 cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is likely required for HIV-1 restriction. SAMHD1 restricts HIV-1 infection in primary MDDCs, and its silencing increases the susceptibility of these cells to HIV-1. SAMHD1 is involved in nucleotide metabolism and may degrade or prevent accumulation of HIV DNA. SAMHD1 and TREX1 are both involved in the cell-intrinsic antiviral response and are associated with Aicardi-Goutières syndrome. SAMHD1 is a key factor in the fate of HIV-1 infection in myeloid cells, and modulating its function could enhance innate and adaptive immune responses. These findings suggest that targeting SAMHD1 could be a strategy for developing dendritic-cell-targeted vaccines against HIV/AIDS.