Cholesterol accumulation is a critical step during the development and progression of atherosclerosis (As). Recently, the expression of Wnt5a has been found to be strikingly upregulated in both murine and human atherosclerotic lesions. However, the effect and mechanism of Wnt5a in atherosclerosis is poorly understood. In the present study, we investigated the effects and potential mechanisms of Wnt5a on cholesterol accumulation during atherosclerosis. We utilized RAW264.7 and vascular smooth muscle cells (VSMCs) treated with ox‐LDL as lipid‐loaded cell models. We found that the expression of Wnt5a protein was increased in a concentration‐dependent and time‐dependent manner under ox‐LDL treatment. To explore the underlying mechanism, we used Wnt5a siRNA to knockdown its expression or applied recombinant Wnt5a (rWnt5a) to stimulate its signaling. After knocking down Wnt5a, the content of total cholesterol (TC) and free cholesterol (FC) increased significantly (P<0.05) upon exposure of the cells to ox‐LDL. Conversely, the content of TC and FC decreased dramatically (P<0.05) after treatment with rWnt5a. More importantly, the expression of Caveolin‐1 and ABCA1 was dramatically reduced upon exposure to ox‐LDL when Wnt5a was knocked down; whereas, in the cells treated with rWnt5a, the expression of these proteins was increased significantly upon the exposure to ox‐LDL. Taken together, these findings demonstrate for the first time that Wnt5a reduces the accumulation of cholesterol in lipid‐loaded cells through regulation of the expression of Caveolin‐1 and ABCA1 at the mRNA levels, which are involved in reverse cholesterol transport. This may present a novel mechanism of Wnt5a‐mediated cholesterol transportation in macrophages and VSMCs. Therefore, targeting the Wnt5a signaling pathway may have clinical implications in atherosclerosis. This article is protected by copyright. All rights reserved.