Abstract Body: Despite decades of improvements, cardiovascular disease remains the top global cause of death worldwide. Cardiovascular mortality has increased over the last 10 years, highlighting the critical need for novel therapies. Despite over 300 genes associated with coronary artery disease (CAD), the leading cause of cardiovascular mortality, mechanisms of how these genes contribute to CAD remain poorly understood. Prior work has shown that the genetically attributable disease risk in CAD lies in smooth muscle cells (SMC), and their associated transition state, fibromyocytes (FMC). Emerging evidence has found that several CAD associated genes influence the ability of SMC to transition to FMCs, suggesting this is a critical mechanism of CAD risk. Here, we describe the novel disease mechanisms of a CAD-associated gene, PR-domain containing 16 (PRDM16), which is enriched in vascular SMCs. First, we demonstrate that genetically higher expression of PRDM16 is associated with higher risk of vascular phenotypes in humans, including CAD, hypertension, ischemic stroke and myocardial infarction. Second, using a SMC-specific knockout of Prdm16 in a lineage traced rodent model of atherosclerosis, we show that loss of Prdm16 leads to a reduced atherosclerotic burden with increased markers of plaque stability. Third, using single cell RNA sequencing, we found that SMC-specific deletion of Prdm16 increased FMCs by over 50% (P=0.004). This was coupled with a near complete shift of native SMCs to those expressing secondary heart field markers (0.976 vs 0.244, P=3.19E-5). Fourth, we found that PRDM16 alters SMC function in vitro, reducing both proliferation and migration. Fifth, we use chromatin accessibility data to identify potential interacting partners with Prdm16 in vivo and discovered strong associations with Ctcf and Pbx1 cofactors. We confirmed that PRDM16 directly binds both CTCF and PBX1 in vitro. Knockdown of either CTCF or PBX1 prevented PRDM16 from reducing SMC proliferation, suggesting that these factors are required for PRDM16 to alter SMC proliferation. Lastly, since PRDM16 is a known epigenetic modifier, we explore whether its epigenetic activity was required to alter SMC function. Inhibiting the epigenetic activity of PRDM16 partially restored the inhibitory effect of PRDM16 on SMC migration but not proliferation. Collectively, these data determine a novel function of PRDM16 on atherosclerosis risk and through its ability to alter phenotypic modulation of SMC.