Atherosclerosis is a major cause of death throughout the world and oxidative stress, which results from the imbalance between reactive species production and antioxidant mechanisms in the arterial wall, is strongly implicated in all stages of this disease. Norbixin is an unusual water-soluble carotenoid found in seeds of Bixa orellana L., a tropical shrub commonly found in Brazil. In this study, we evaluated whether norbixin could attenuate oxidative stress in the aortic tissue during experimental atherosclerosis. New Zealand rabbits (CEUA/UFSM n 28/2010) received regular chow or hypercholesterolemic diet alone (regular chow + 0.5% cholesterol) or enriched with norbixin 10, 30, or 100 mg/kg for 60 days. The atherogenic diet increased the non-protein thiol groups (NPSH) content and superoxide dismutase (SOD), thioredoxin reductase (TrxR), catalase (CAT), and glutathione reductase (GR) activities in the aortic tissue compared with the control group (45.5 ± 5.6 vs. 32.6±4.3 nmol/mg protein; 1.27 ± 0.05 vs. 0.90 ± 0.04 U/mg protein; 27.5 ± 3.8 vs. 15.8 ± 2.1 nmol DTNB reduced/min/mg protein, 1.8 ±0.3 vs. 1.2±0.2 K/mg protein; 4.8±1.07 vs. 1.5±0.3 nmol NADPH/min/g protein, respectively, p<0.05). The increased levels of NPSH were completely restored by all norbixin doses (26.8 - 31.8 nmol/mg protein). Norbixin at 10 mg/kg completely prevented the increase in CAT activity (1.0 ± 0.1 K/mg protein) but partially prevented such increase at 30 and 100 mg/kg (1.5 ± 0.1 and 1.4 ± 0.2 K/mg protein). The alteration in SOD, TrxR, and GR activities were completely prevented by all norbixin doses (0.8 – 0.9 U/mg protein, 13.9 – 17.1 nmol DTNB reduced/min/mg protein, and 2.4 - 2.8 nmol NADPH/min/mg protein, respectively). Glutathione peroxidase activity was not affected by atherogenic diets or norbixin treatment. Our results indicate that norbixin plays a beneficial role against changes in intracellular enzymatic and non-enzymatic antioxidant defenses induced by hypercholesterolemia.