Paeonol, a potent antioxidant isolated from cortex moutan, possesses athero‑protective activity, yet the detailed mechanisms are not fully investigated. This study was conducted to explore the role of paeonol and its underlying mechanisms in RAW264.7 macrophages and apolipoprotein E‑deficient (ApoE(‑/‑)) mice. Paeonol treatment significantly attenuated intracellular lipid accumulation in macrophages, which may be the result of decreased oxidized low‑density lipoprotein (ox‑LDL) uptake and increased cholesterol efflux. Additionally, paeonol markedly inhibited the mRNA and protein expression of the cluster of differentiation 36 (CD36) by decreasing nuclear translocation of c‑Jun [a subunit of activator protein‑1 (AP‑1)]. Moreover, paeonol upregulated the protein stability of ATP‑binding cassette transporter A1 (ABCA1) by inhibiting calpain activity, while ABCA1 mRNA expression was not altered. Furthermore, small hairpin RNA (shRNA) targeting haem oxygenase‑1 (HO‑1) inhibited the paeonol‑mediated beneficial effects on the expression of c‑Jun, CD36, ABCA1, calpain activity and lipid accumulation in macrophages. Accordingly, paeonol retarded the progress of atherosclerosis in ApoE(‑/‑) mice and modulated the expression of CD36 and ABCA1 in aortas similarly to that observed in macrophages. These results indicate that paeonol provides protective effects on foam cell formation by a novel HO‑1‑dependent mediation of cholesterol efflux and lipid accumulation in macrophages.