SAMHD1 is a dGTP-activated dNTPase that has been implicated as a modulator of the innate immune response. In monocytes and their differentiated derivatives, as well as in quiescent cells, SAMHD1 strongly inhibits HIV-1 infection and, to a lesser extent, HIV-2 and simian immunodeficiency virus (SIV) because of their virion-associated virulence factor Vpx, which directs SAMHD1 for proteasomal degradation. Here, we used a combination of biochemical and virologic approaches to gain insights into the functional organization of human SAMHD1. We found that the catalytically active recombinant dNTPase is a dGTP-induced tetramer. Chemical cross-linking studies revealed SAMHD1 tetramers in human monocytic cells, in which it strongly restricts HIV-1 infection. The propensity of SAMHD1 to maintain the tetrameric state in vitro is regulated by its C terminus, located outside of the catalytic domain. Accordingly, we show that the C terminus is required for the full ability of SAMHD1 to deplete dNTP pools and to inhibit HIV-1 infection in U937 monocytes. Interestingly, the human SAMHD1 C terminus contains a docking site for HIV-2/SIVmac Vpx and is known to have evolved under positive selection. This evidence indicates that Vpx targets a functionally important element in SAMHD1. Together, our findings imply that SAMHD1 tetramers are the biologically active form of this dNTPase and provide new insights into the functional organization of SAMHD1.