Background and Purpose- Genome-wide association studies have identified the HDAC9 (histone deacetylase 9) gene region as a major risk locus for atherosclerotic stroke and coronary artery disease in humans. Previous results suggest a role of altered HDAC9 expression levels as the underlying disease mechanism. rs2107595, the lead single nucleotide polymorphism for stroke and coronary artery disease resides in noncoding DNA and colocalizes with histone modification marks suggestive of enhancer elements. Methods- To determine the mechanisms by which genetic variation at rs2107595 regulates HDAC9 expression and thus vascular risk we employed targeted resequencing, proteome-wide search for allele-specific nuclear binding partners, chromatin immunoprecipitation, genome-editing, reporter assays, circularized chromosome conformation capture, and gain- and loss-of-function experiments in cultured human cell lines and primary immune cells. Results- Targeted resequencing of the HDAC9 locus in patients with atherosclerotic stroke and controls supported candidacy of rs2107595 as the causative single nucleotide polymorphism. A proteomic search for nuclear binding partners revealed preferential binding of the E2F3/TFDP1/Rb1 complex (E2F transcription factor 3/transcription factor Dp-1/Retinoblastoma 1) to the rs2107595 common allele, consistent with the disruption of an E2F3 consensus site by the risk allele. Gain- and loss-of-function studies showed a regulatory effect of E2F/Rb proteins on HDAC9 expression. Compared with the common allele, the rs2107595 risk allele exhibited higher transcriptional capacity in luciferase assays and was associated with higher HDAC9 mRNA levels in primary macrophages and genome-edited Jurkat cells. Circularized chromosome conformation capture revealed a genomic interaction of the rs2107595 region with the HDAC9 promoter, which was stronger for the common allele as was the in vivo interaction with E2F3 and Rb1 determined by chromatin immunoprecipitation. Gain-of-function experiments in isogenic Jurkat cells demonstrated a key role of E2F3 in mediating rs2107595-dependent transcriptional regulation of HDAC9. Conclusions- Collectively, our findings imply allele-specific transcriptional regulation of HDAC9 via E2F3 and Rb1 as a major mechanism mediating vascular risk at rs2107595.