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The cellular insulator protein CTCF plays a role in HSV-1 latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the LAT enhancer was bound by CTCF during latency and underwent CTCF eviction at early times post-reactivation in mice latent with 17syn+. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (ΔCTRL2) in the 17syn+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells, but not neuronal cells. Following ocular infection, mouse survival decreased in the ΔCTRL2 cohort and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with ΔCTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the ganglia infected with ΔCTRL2 by 31 days post-infection, relative to 17syn+, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, ChIP-seq analyses of TG from ΔCTRL2 infected mice confirmed that the distribution of the repressive H3K27me3 mark on the ΔCTRL2 recombinant genomes was altered compared to the wild-type, indicating that CTRL2 site modulates the repression of IE genes during latency. [ABSTRACT FROM AUTHOR]