The cHS4 chromatin insulator has been shown to improve the expression of integrating gene transfer vectors by reducing the impact of silencing chromosomal position effects. To better understand the underlying mechanisms of this protection, we investigated the influence of this element on the epigenetic modifications of a gammaretroviral reporter vector. In HT1080 cells, we found that a fourfold increase in the level of green fluorescent protein (GFP) reporter expression from the cHS4-insulated vector was correlated with a twofold increase in acetylation at lysines 9 and 14 of histone H3, but not with CpG methylation. In a mouse bone marrow transduction and transplantation model, we found that a 10-fold increase in the likelihood of GFP expression from the cHS4-insulated vector was correlated with an eightfold increase in histone H3 acetylation, as well as a fourfold decrease in CpG methylation. Histone hyperacetylation peaked at the cHS4 core, and in vivo diminished nearly threefold through the central portion of the vector. Taken together, these studies demonstrate that the cHS4 chromatin insulator reduces gammaretroviral vector silencing by modulating epigenetic modifications of integrated provirus, and identify a specific topological distribution of these modifications that may prove informative for future vector designs.