Atherosclerosis (AS) is a chronic inflammatory disease threatening human health, and vascular smooth muscle cells (VSMCs) are involved in AS processes. Baicalin is a flavonoid compound, which has anti-atherosclerotic effect. The aim of our study was to explore the molecular mechanism of baicalin on AS. The expression of miR-126-5p was measured in peripheral blood of AS patients and healthy control. We found miR-126-5p expression was decreased in AS. Then, high-mobility group box 1 (HMGB1) was verified as a target of miR-126-5p and its expression was increased in AS. Similarly, miR-126-5p and HMGB1 expression was downregulated and upregulated in oxidized low-density lipoprotein treated VSMCs (ox-LDL-VSMCs), respectively. Furthermore, baicalin upregulated miR-126-5p and downregulated HMGB1 expression. Functionally, baicalin significantly inhibited ox-LDL-VSMCs proliferation and migration, and miR-126-5p targets HMGB1 to enhance the inhibition induced by baicalin. Taken together, baicalin is able to prevent AS, which suppressed the proliferation and migration of ox-LDL-VSMCs through upregulating miR-126-5p by targeting HMGB1. These findings suggested that baicalin is an effective drug to alleviate AS, and miR-126-5p is a novel therapeutic target for AS.