Monocyte-derived macrophages (MDMs) are an important target for HIV-1 despite SAMHD1, a myeloid restriction factor for which HIV-1 lacks a counteracting accessory protein. The antiviral activity of SAMHD1 is modulated by phosphorylation of T592 by cyclin-dependent kinases (CDK). We show that treatment of MDMs with neocarzinostatin, a compound that introduces double strand breaks (DBS) in genomic DNA, results in the decrease of phosphorylated SAMHD1, activating its antiviral activity and blocking HIV-1 infection. The effect was specific for DSB as DNA damage induced by UV light irradiation did not affect SAMHD1 phosphorylation and did not block infection. The block to infection was at reverse transcription and was counteracted by Vpx, demonstrating that it was caused by SAMHD1. Neocarzinostatin treatment also activated an innate immune response that induced interferon-stimulated genes but this was not involved in the block to HIV-1 infection, as it was not relieved by an interferon-blocking antibody. In response to Neocarzinostatin-induced DNA damage, the level of the CDK inhibitor p21cip1 increased which could account for the decrease of phosphorylated SAMHD1. The results show that the susceptibility of MDMs to HIV-1 infection can be affected by stimuli that alter the phosphorylation state of SAMHD1, one of which is the DNA damage response.