Abstract Background Numerous genome-wide association studies revealed that SNPs at phosphatase and actin regulator 1 (PHACTR1) locus are strongly correlated with coronary artery disease (CAD). However, the mechanism linking these variants to CAD remains uncertain. Purpose We studied the biological functions and molecular mechanisms of PHACTR1 in atherosclerosis. Methods and results Analysis of GTEx database showed that CAD-related SNPs in PHACTR1 are cis-eQTLs for PHACTR1 in arteries. Therefore, we generated Phactr1 knockout mice and crossed them with apolipoprotein E-deficient (ApoE−/−) mice to induce atherosclerosis by high-fat/high-cholesterol (HF-HC) diet. Phactr1 deficiency significantly inhibited atherosclerosis with decreased inflammatory cell infiltration. Western blot showed that PHACTR1 was restricted to endothelial cells (ECs) in mice. Mechanistically, RNAseq of aortic ECs revealed that the major molecular function of PHACTR1 was transcriptional regulation. PPARγ/RXRα was the top transcription factor, and PPARγ target gene expression substantially increased in Phactr1−/− mice. Moreover, we generated endothelial cell specific Phactr1−/−, ApoE−/− mice and found decreased atherosclerotic plaque area in aortic sinus. In vitro, PHACTR1 associated with PPARγ and inhibited PPARγ transcriptional activity. The inhibitory effect of PHACTR1 on PPARγ required its shuttling from cytosol to nucleus triggered by disturbed flow, a well-established pro-atherosclerotic stimulus. Conclusion Our results identified PHACTR1 as a mechanosensitive corepressor of PPARγ in ECs to promote atherosclerosis. Endothelial PHACTR1 is a potential therapeutic target for atherosclerosis treatment. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China (NSFC), China Postdoctoral Science Foundation (CPSF)