Polyphenols can easily react with reactive oxygen species (ROS) resulting in powerful antioxidant activity. Pyrogallol is a polyphenol found in a variety of fruits and vegetables. Therefore, plants containing pyrogallol can be used for their antioxidant, anti-bacterial, anti-fungal, anti-allergic, and anti-inflammatory properties. However, its effectiveness as an antioxidant is still a matter of debate because pyrogallol is also a superoxide anion generator and induces superoxide anion-mediated death of several types of cells. In this study, we tried to confirm the antioxidant effects and mechanisms of pyrogallol in mouse neuroblastoma (N2A), human neural stem cells (F3), and mouse brain. Treatments with various concentrations of pyrogallol significantly increased the free radical scavenging ability and reduced lipid peroxidation in N2A and F3 cells in a concentration-dependent manner. In the cytotoxicity test, low concentrations of pyrogallol (0–10 µg/mL) did not significantly influence cell viability, while high concentrations (125-1000 µg/mL) induced death in N2a and F3 cells. Furthermore, treatment with pyrogallol(1.0, 3.2, and 10.0 µg/mL) up-regulated the mRNA expression of superoxide dismutases 1, 2, and 3, glutathione peroxidase (GPx)1, and phospholipid hydroperoxide GPx in N2A and F3 cells, in the brains of mice that were orally administered pyrogallol (1.0, 3.2, and 10.0 mg/kg/day) for 7 days. The mice did not exhibit any toxic effects due to this treatment. Taken together, although pyrogallol induced cell death at high concentrations, it might be a good candidate antioxidant for neurodegenerative disease at low concentrations.