This study was conducted to investigate the effects of Opuntia humifusa supplementation on lipid peroxidation and superoxide dismutase (SOD) protein expression at resting state in various organs of rats fed a high-fat diet. Sixteen Sprague-Dawley male rats, 6 weeks of age, were randomly divided into two groups: a control diet group (CG, n=8) and an experimental diet group (EG, n=8). They were given a high-fat diet (CG) or a diet supplemented with 5% of O. humifusa (EG) for 8 weeks. The results showed that the malondialdehyde (MDA) levels of the kidney and the liver were significantly lower in the EG group than in the CG group (p<0.01). In addition, the MDA levels in the skeletal muscle of the EG group tended to be lower than those in the CG group, but this difference was not significant. The Cu, Zn-SOD protein expression in the kidney of the EG group was significantly increased compared with that of the CG group (p<0.01). The Mn-SOD protein expression in the skeletal muscle of the EG group was significantly increased compared with that of the CG group (p<0.01). These results suggest that O. humifusa supplementation has antioxidative properties, which are exerted in a specific organ manner, and that it inhibits the action of lipid peroxidation and the expression of SOD in rats fed a high-fat diet.