SAMHD1 is a host protein responsible, at least in part, for the inefficient infection of dendritic, myeloid, and resting T cells by HIV-1. Interestingly, HIV-2 and SIVsm viruses are able to counteract SAMHD1 by targeting it for proteasomal degradation using their Vpx proteins. It has been proposed that SAMHD1 is a dGTP-dependent deoxynucleoside triphosphohydrolase (dNTPase) that restricts HIV-1 by reducing cellular dNTP levels to below that required for reverse transcription. However, nothing is known about SAMHD1 posttranslational modifications and their potential role in regulating SAMHD1 function. We used (32)P labeling and immunoblotting with phospho-specific antibodies to identify SAMHD1 as a phosphoprotein. Several amino acids in SAMHD1 were identified to be sites of phosphorylation using direct mass spectrometry. Mutation of these residues to alanine to prevent phosphorylation or to glutamic acid to mimic phosphorylation had no effect on the nuclear localization of SAMHD1 or its sensitivity to Vpx-mediated degradation. Furthermore, neither alanine nor glutamic acid substitutions had a significant effect on SAMHD1 dNTPase activity in an in vitro assay. Interestingly, however, we found that a T592E mutation, mimicking constitutive phosphorylation at a main phosphorylation site, severely affected the ability of SAMHD1 to restrict HIV-1 in a U937 cell-based restriction assay. In contrast, a T592A mutant was still capable of restricting HIV-1. These results indicate that SAMHD1 phosphorylation may be a negative regulator of SAMHD1 restriction activity. This conclusion is supported by our finding that SAMHD1 is hyperphosphorylated in monocytoid THP-1 cells under nonrestrictive conditions.