Glutathione is a well-known chemotherapeutic agent and a popular nutritional supplement for liver disease and oxidative stress. Our previous studies reported the suppressive effects of the glutathione-enriched Saccharomyces cerevisiae FF-8 (FF-8) strain on carbon tetrachloride- and alcohol-induced oxidative stress in rats. The purpose of the current study was to investigate the effect of the FF-8 strain on lipid peroxidation in tissues of rats with orotic acid (OA)-induced fatty liver. OA treatment showed a significant decrease in body weight gain compared to the normal diet, and simultaneous addition of FF-8 and OA had the same effect. OA treatment produced an increase in liver weight, however, this also increased with simultaneous addition of FF-8 and OA. Liver lipid peroxidation was significantly increased by OA, but was significantly decreased by FF-8 strain treatment. This same tendency was found in the kidney and heart. Concentration levels of hepatic glutathione and zinc are known to be closely associated with the antioxidant system, and OA treatment led to reductions in liver glutathione and zinc concentrations, whereas these were significantly increased by FF-8 strain treatment in OA feeding rats. These results suggest that the glutathione-enriched S. cerevisiae FF-8 strain may positively mediate orotic acid-induced oxidative stress by enhancing glutathione and zinc levels in rat livers.