Intrinsic antiviral host factors are proteins that can confer cellular defense by limiting virus replication. Viruses hijack ubiquitin machinery to modify the cellular proteome to subvert these host defenses. The herpes simplex virus 1 (HSV-1) expresses ICP0, which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify new restriction factors antagonized by ICP0, we compared the proteomes associated with viral DNA (vDNA) during HSV-1 infection with wild-type (WT) virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen 5 (SLFN5) as a new ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5 which leads to its proteasomal degradation. In the absence of ICP0, we demonstrate SLFN5 binds vDNA to represses HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results identify SLFN5 as a new restriction factor that inhibits viral transcription and document the first viral countermeasure reported to overcome SLFN antiviral activity.