Mesenchymal stem cells (MSCs) have emerged as a cell-based therapy in many diseases including atherosclerosis (AS) due to their capability of immunomodulation and tissue regeneration. However, the pathway for MSCs' antiatherosclerotic activity remains to be elucidated. Here, we test the hypothesis that microRNA-221 (miR-221) from MSC-derived extracellular vesicles (EVs) alleviates AS. Male ApoE-/- mice were fed a high-fat diet for 12 weeks to induce AS, and were then treated with human bone marrow mesenchymal stem cell-derived EVs by tail vein injection. The expression pattern of miR-221 and N-acetyltransferase-1 (NAT1) in AS mice was characterized by quantitative RNA analysis and their interaction was identified by dual-luciferase reporter gene assay. In other studies, human arterial smooth muscle cells treated with oxidized low-density lipoprotein-were co-cultured with MSC-released EVs to evaluate the EV-mediated transfer of miR-221. NAT1 was highly expressed in atherosclerotic lesions. Adenovirus-mediated NAT1 knockdown resulted in a reduced lipid deposition in AS mice. Human bone marrow mesenchymal stem cell -derived EVs carrying miR-221 were internalized by human arterial smooth muscle cells and transferred their miR-221 contents to downregulate the target gene NAT1. Injection of miR-221-containing EVs inhibited lipid deposition in AS mice, in part by downregulating NAT1. The present study provides evidence that miR-221 shuttled by MSC-derived EVs can inhibit atherosclerotic plaque formation in AS model mice, suggesting that miR-221 may serve as a target for improving MSC-based therapeutic strategy against AS. Copyright © 2020 Elsevier Inc. All rights reserved.