High-density lipoprotein (HDL) from healthy subjects (HDLhealthy) has anti-inflammatory effects, whereas HDL from patients with coronary artery disease (HDLCAD) is functionally impaired, which may affect atherosclerosis formation differently. We previously demonstrated that HDLhealthy inhibits the expression of the long noncoding RNA high-density lipoprotein-regulated angiogenesis in coronary artery disease (HDRACA), whereas HDLCAD is much less effective. HDRACA is highly expressed in endothelial cells (ECs) of arteriosclerosis obliterans and is associated with the expression of adhesion molecules and chemokines. However, whether HDLhealthy and HDLCAD affect vascular inflammation and atherosclerosis by regulating HDRACA expression remains unclear. Here, we found that HDLhealthy suppressed HDRACA expression in ECs, thereby inhibiting endothelial chemotactic and adhesive effects, whereas HDLCAD had less of an effect. Mechanistically, HDRACA bound to heat shock protein β-1 (HSPB1), an anti-inflammatory protein that underwent liquid-liquid phase separation (LLPS) mediated by its N-terminal domain (NTD) and C-terminal domain (CTD). Overexpression of HDRACA promoted LLPS of HSPB1, inhibited HSPB1-IKKβ interaction, and activated the NF-ĸB pathway, leading to increased expression of adhesion molecules and chemokines. HDLhealthy enhanced the phosphorylation of serine residues 15, 78, and 82 in the NTD of HSPB1 to inhibit its LLPS by suppressing HDRACA expression, whereas HDLCAD was less effective. Delivering HDRACA into mouse aortic ECs also promoted LLPS of HSPB1, leading to enhanced vascular inflammation and accelerated atherosclerosis formation in low-density lipoprotein receptor null mice. Our findings identified HDRACA-induced LLPS of HSPB1 as a novel mechanism linking HDL dysfunction and atherosclerosis development. HDLhealthy suppressed HDRACA-induced LLPS of HSPB1 to inhibit vascular inflammation and exerted anti-atherosclerotic effects, whereas HDLCAD lost its protective role because of its inefficacy in HDRACA inhibition. These findings suggest that improving HDL function, inhibiting HDRACA, and blocking LLPS of HSPB1 may represent promising therapeutic strategies for atherosclerosis.