The root of Polygala radix has been widely known as an oriental traditional medicinal stuff that improves memory. However, its mechanism of action remains unclear. In this study, the effect of Polygala radix hot water extracts (PRHWE) on cognitive function related to the activity of acetylcholinesterase (AchE) derived from neural cells (PC12) in addition to antioxidant activity was examined both in a cell-free system and live cells. First, in the study on cell viability using an MTT assay, PRHWE did not exhibit any cell toxicity at 0.1% (w/v) or below. It also was observed that PRHWE increased the scavenging activity of DPPH radical, hydrogen peroxide and superoxide, reducing power in a dose-dependent manner. In particular, PRHWE had a protective effect on DNA oxidation induced by hydroxyl radicals. Additionally, it inhibited the production of inducible nitric oxide in neuronal cells. Furthermore, the AchE activity decreased with increasing concentrations. In addition, PRHWE increased the expression level of SOD-1 and NOS-2 in PC12 cells. Moreover, the transcriptional activities of p53 and NF-${kappa}B$ were reduced in the presence of PRHWE in an experiment using a reporter gene assay. Therefore, these results prove that PRHE has antioxidative and protective effects on neuronal cells, suggesting that it may have great potential as a therapeutic agent for human health.