Hesperidin, a citrus bioflavonoid, exerts numerous pharmacological activities. However, its protective effect against atherosclerosis in vivo remains poorly understood. In the present study, we aimed to observe the effects of hesperidin on high fat diet (HFD)-induced atherosclerosis using LDL receptor deficient (LDLr-/-) mice. After 12 weeks of treatment, the animals were sacrificed. The blood samples were collected for further analysis. Mouse peritoneal macrophages were collected. Hepatic lipid content, quantification of atherosclerosis, assessment of oxidative stress and inflammation, gene expressions were performed on liver and aorta samples. The data showed that hesperidin ameliorated HFD-induced weight gain, improved insulin resistance and ameliorated hyperlipidemia. Hesperidin suppressed HFD-induced hepatic steatosis, atherosclerotic plaque area and macrophage foam cell formation. Further study showed that hesperidin down-regulated expressions of acetyl coenzyme A carboxylase alpha (ACCα) and fatty acid synthase (FAS) which are two key enzymes in fatty acid and triglyceride synthesis in liver; and upregulated expression of hepatic ATP-binding cassette transporters G8 (ABCG8), macrophage ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) which are transporters involved in the process of reverse cholesterol transport. Hesperidin also reduced oxidative stress by normalizing activities of antioxidant enzymes and inflammation in HFD-fed LDLr−/− mice. These findings suggest that hesperidin reduced atherosclerosis via its pleiotropic effects, including improvement of insulin resistance, amelioration of lipid profiles, inhibition of macrophage foam cell formation, anti-oxidative effect and anti-inflammatory action.