Nucleoside reverse transcriptase inhibitors (NRTIs) are a key component of highly active anti-retroviral therapy. Once NRTIs are phosphorylated to a triphosphate form in the cell (NRTI-TPs), they directly compete with deoxynucleotide triphosphates (dNTPs) for the active site of reverse transcriptase (RT).Therefore, the cellular concentration of dNTPs in infected cells directly impacts NRTI efficacy.Since SAM domain and HD domain containing protein 1 (SAMHD1), a host restriction factor of HIV/SIV, is able to hydrolyze cellular dNTPs into dNs and maintain low dNTP concentrations in non-dividing cells such as macrophages, we tested whether SAMHD1 alters HIV-1 sensitivity to NRTIs.This could occur by SAMHD1 mediated modulation of cellular dNTP concentrations or by direct hydrolysis of NRTI-TPs to an inactive form.To test this, drug efficacy was measured in HIV-1 transduced THP-1 cells with shRNA targeting SAMHD1.Knockdown of SAMHD1 increased cellular dNTP levels and decreased HIV-1 sensitivity to AZT.Additionally, primary human macrophages and activated CD4+ T cells were treated with virus-like particles (VLPs) expressing Vpx, the accessory protein of HIV-2/SIV, which targets SAMHD1 for proteasomal degradation. SAMHD1 degradation in macrophages significantly reduced HIV-1 sensitivity to NRTIs and reduced sensitivity to a lesser extent in CD4+ T cells. We also showed with HPLCthat SAMHD1 is not able to hydrolyze AZT-TP and dideoxynucleotide triphosphates (ddNTPs) to their nucleoside derivatives, suggesting that SAMHD1 does not impact the concentration of the active form of NRTIs in cells.Interestingly, we showed that ddGTP and GTP can allosterically activate SAMHD1 providing further insight into the specificity of the allosteric site and regulation of the enzyme.This study demonstrates that degradation of SAMHD1 decreases HIV-1 sensitivity to NRTIs, primarily in macrophages; by altering cellular dNTP concentrations and that NRTI-TPs are not subject to SAMHD1-mediated hydrolysis.