Atherosclerosis, the underlying cause of most heart attacks and strokes, results from lipid accumulation in cells of the artery wall. Gene therapy, delivered directly to the artery wall, has the potential to prevent and reverse atherosclerosis. However, lipid accumulates primarily in cells below the endothelium, which are difficult to reach with gene therapy vectors. Our goal is to remove lipid from these cells by delivering therapeutic microRNA (miR) that increases cholesterol export from the cells. We hypothesized that if we introduced a therapeutic gene expressing the miR to endothelial cells (cells along the artery lumen), the endothelial cells would release this miR (anti-miR-33a-5p) via extracellular vesicles (exosomes) that transport miR between neighboring cells. We also hypothesized that smooth muscle cells (SMC) and macrophages would take up the therapeutic miR-containing exosomes, leading to higher expression of a critical cholesterol export protein (ABCA1) and increased cholesterol export. To test this in vitro, we introduced a therapeutic gene encoding the miR into endothelial cells and used RT-qPCR to test if the miR was released into the endothelial cell culture medium (CM) via exosomes. After confirming the presence of the therapeutic miR in exosomes purified from CM, we treated SMC and macrophages by incubating the cells with the exosome-containing CM. After incubation, we measured ABCA1 protein expression and cholesterol export. Expression of ABCA1 protein increased by 1.6- and 2.2-fold in SMC and macrophages, respectively, while cholesterol export increased by 1.4- and 1.6-fold. We conclude that gene therapy delivered to endothelial cells can produce therapeutic miR that is transferred to neighboring artery wall cells via exosomes, and increases cholesterol export in these target cells. If also effective in vivo, our approach has potential for reducing the severity of atherosclerosis by delivering therapeutic miR to cells that are difficult to reach with gene therapy.