An ethanol extract of the stem of Opuntia ficus-indica var. saboten (OFS) was assessed to determine the mechanism(s) of its antioxidant activity. The ethanol extract exhibited a concentration-dependent inhibition of linoleic acid oxidation in a thiocyanate assay system. In addition, the OFS extract showed dose-dependent free-radical scavenging activity, including DPPH radicals, superoxide anions (O(2)(*-)), and hydroxyl radicals (*OH), using different assay systems. The OFS ethanol extract was also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals in a Fenton's reaction mixture. Furthermore, the extract showed significant (p < 0.01) dose-dependent protection of mouse splenocytes against glucose oxidase-mediated cytotoxicity. Finally, the OFS extract was characterized as containing a high amount of phenolics (180.3 mg/g), which might be the active compounds responsible for the antioxidant properties of the OFS extract.