We investigated the protective effects of Glycyrrhizae Radix extract against peroxynitrite (ONOO-)-induced oxidative stress under in vivo as well as in vitro conditions. The extract showed strong ONOO- and nitric oxide (NO) scavenging effects under in vitro system, in particular higher activity against ONOO-. Furthermore, elevations of plasma 3-nitrotyrosine levels, indicative of in vivo ONOO- generation and NO production, were shown using a rat in vivo ONOO(-)-generation model of lipopolysaccharide injection plus ischemia-reperfusion. The administration of Glycyrrhizae Radix extract at doses of 30 and 60 mg/kg body weight/day for 30 days significantly reduced the concentrations of 3-nitrotyrosine and NO and decreased inducible NO synthase activity. In addition, the nitrated tyrosine protein level and myeloperoxidase activity in the kidney were significantly lower in rats given Glycyrrhizae Radix extract than in control rats. However, the administration of Glycyrrhizae Radix extract did not result in either significant elevation of glutathione levels or reduction of lipid peroxidation in renal mitochondria. Moreover, the in vivo ONOO- generation system resulted in renal functional impairment, reflected by increased plasma levels of urea nitrogen and creatinine, whereas the administration of Glycyrrhizae Radix extract reduced these levels significantly, implying that the renal dysfunction induced by ONOO- was ameliorated. The present study suggests that Glycyrrhizae Radix extract could protect the kidneys against ONOO- through scavenging ONOO- and/or its precursor NO, inhibiting protein nitration and improving renal dysfunction caused by ONOO-.