CXC chemokine ligand 12 (CXCL12) is a member of the CXC chemokine family and mainly acts on cell chemotaxis. CXCL12 also elicits a proatherogenic role, but the molecular mechanisms have not been fully defined yet. We aimed to reveal if and how CXCL12 promoted atherosclerosis via regulating lipid metabolism. In vitro, our data showed that CXCL12 could reduce ABCA1 expression, and it mediated cholesterol efflux from THP-1-derived macrophages to apoA-I. Data from the luciferase reporter gene and chromatin immunoprecipitation assays revealed that transcription factor 21 (TCF21) stimulated the transcription of ABCA1 via binding to its promoter region, which was repressed by CXCL12. We found that CXCL12 increased the levels of phosphorylated glycogen synthase kinase 3β (GSK3β) and the phosphorylation of β-catenin at the Thr120 position. Inactivation of GSK3β or β-catenin increased the expression of TCF21 and ABCA1. Further, knockdown or inhibition of CXC chemokine receptor 4 (CXCR4) blocked the effects of CXCL12 on TCF21 and ABCA1 expression and the phosphorylation of GSK3β and β-catenin. In vivo, the overexpression of CXCL12 in Apoe mice via lentivirus enlarged the atherosclerotic lesion area and increased macrophage infiltration in atherosclerotic plaques. We further found that the overexpression of CXCL12 reduced the efficiency of reverse cholesterol transport and plasma HDL-C levels, decreased ABCA1 expression in the aorta and mouse peritoneal macrophages (MPMs), and suppressed cholesterol efflux from MPMs to apoA-I in Apoe mice. Collectively, these findings suggest that CXCL12 interacts with CXCR4 and then activates the GSK-3β/β-catenin/TCF21 signaling pathway to inhibit ABCA1-dependent cholesterol efflux from macrophages and aggravate atherosclerosis. Targeting CXCL12 may be a novel and promising strategy for the prevention and treatment of atherosclerotic cardiovascular diseases.