BACKGROUND AND AIMS Cholesterol-loaded smooth muscle cells (SMCs) modify their phenotypic behavior becoming foam cells. To characterize the role of ABCA1 and HDL3 in this process, we evaluated HDL3 effects on cholesterol-induced phenotypic changes in SMCs expressing or not ABCA1. METHODS SMCs, isolated from the aortae of wild-type (WT) and Abca1 knock-out (KO) mice, were cholesterol-loaded using a "water-soluble cholesterol''. RESULTS Cholesterol loading downregulates the expression of Acta2 and calponin (SMC markers), and increases the expression of Mac-2, CD11b and MHCII (inflammation-related genes and surface antigens) and Abca1, Abcg1. HDL3 normalizes SMC marker expression and reduces the expression of inflammation-related genes/proteins in WT cells, an effect not observed with free apoA-I. The effect of HDL3 is almost lost in Abca1 KO cells, as well as when Abca1 is silenced in WT SMC. HDL3 does not differently affect cholesterol downloading in WT or KO cells and stimulates phospholipids removal in WT cells. Similarly, the expression of myocardin and its modulators, such as miR-143/145, is reduced by cholesterol loading in WT and Abca1 KO SMCs; HDL3 normalizes their levels in WT cells but not in KO cells. On the contrary, cholesterol loading induces Klf4 expression while HDL3 restores Klf4 to basal levels in WT cells, but again this effect is not observed in KO cells. CONCLUSIONS Our results indicate that HDL3, by interacting with ABCA1, modulates the miR143/145-myocardin axis and prevents the cholesterol-induced gene expression modification in SMCs regardless of its cholesterol unloading capacity.