Atherosclerosis is a chronic inflammatory disease related to a massive accumulation of cholesterol in the artery wall. Photobiomodulation therapy (PBMT) has been reported to possess cardioprotective effects but has no consensus on the underlying mechanisms. Here, we aimed to investigate whether PBMT could ameliorate atherosclerosis and explore the potential molecular mechanisms. The Apolipoprotein E (ApoE)−/− mice were fed with western diet (WD) for 18 weeks and treated with PBMT once a day in the last 10 weeks. Quantification based on Oil red O‐stained aortas showed that the average plaque area decreased 8.306 ± 2.012% after PBMT (P < .05). Meanwhile, we observed that high‐density lipoprotein cholesterol level in WD + PBMT mice increased from 0.309 ± 0.037 to 0.472 ± 0.038 nmol/L (P < .05) compared with WD mice. The further results suggested that PBMT could promote cholesterol efflux from lipid‐loaded primary peritoneal macrophages and inhibit foam cells formation via up‐regulating the ATP‐binding cassette transporters A1 expression. A contributing mechanism involved in activating the phosphatidylinositol 3‐kinases/protein kinase C zeta/specificity protein 1 signalling cascade. Our study outlines that PBMT has a protective role on atherosclerosis by promoting macrophages cholesterol efflux and provides a new strategy for treating atherosclerosis.