Objectives Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in carotid artery stenosis. The purpose of this study was to investigate the diagnostic value of serum miR-28-5p in asymptomatic carotid artery stenosis and its regulation on the proliferation and migration of VSMCs. Methods Serum miR-28-5p levels in 65 healthy controls and 68 asymptomatic carotid artery stenosis patients were detected by qRT–PCR. The receiver-operating characteristic curve was applied to elucidate the diagnostic value of serum miR-28-5p for carotid artery stenosis patients. The specificity of miRNA targets was detected by luciferase reporter assay. CCK-8 and Transwell assay were applied to detect proliferation and migration of cells. Pearson correlation test was used to investigate the correlation between Forkhead box subclass O 1 (FOXO1) and serum miR-28-5p. Results Serum miR-28-5p was significantly reduced in asymptomatic carotid artery stenosis patients. Moreover, miR-28-5p could distinguish asymptomatic carotid artery stenosis patients from healthy controls, with sensitivity and specificity of 86.8% and 81.5%, respectively, indicating its high diagnostic value. The overexpression of miR-28-5p inhibited the proliferation and migration of VSMCs, while inhibition of miR-28-5p resulted in the opposite effect. What is more, FOXO1, a direct target of miR-28-5p, was significantly increased in asymptomatic carotid artery stenosis patients. Inhibition of miR-28-5p in VSMCs reversed the reduction of FOXO1 levels in patients. Conclusions miR-28-5p is a valuable diagnostic biomarker for asymptomatic carotid artery stenosis and can affect the proliferation and migration of VSMCs by regulating FOXO1.