Methylglyoxal is a metabolite of glucose. Since serum methylglyoxal level is increased in diabetic patients, methylglyoxal is implicated in diabetic complications such as cognitive impairment. This study aimed to evaluate the effects of tenuigenin, an active component of roots of Polygala tenuifolia Willdenow, on methylglyoxal-induced cell injury in a primary culture of rat hippocampal neurons. MTT and Hoechst 33342 staining, together with flow cytometric analysis using annexin-V and propidium (PI) label, indicated that tenuigenin pretreatment attenuated methylglyoxal -induced apoptotic cell death in primary cultured hippocampal neurons, showing a dose-dependent pattern. Furthermore, 2, 7-dichlorodihydrofluorescein diacetate was used to detect the level of intracellular reactive oxygen species. Tenuigenin decreased the elevated reactive oxygen species induced by methylglyoxal. In addition, tenuigenin inhibited activation of caspase-3 and reversed down-regulation of the ratio of Bcl-2/Bax, both of which were induced by methylglyoxal stimulation. The results suggest that tenuigenin displays antiapoptotic and antioxidative activity in hippocampal neurons due to scavenging of intracellular reactive oxygen species, regulating Bcl-2 family and suppressing caspase-3 activity induced by methylglyoxal, which might explain at least in part the beneficial effects of tenuigenin against degenerative disorders involving diabetic cognitive impairment.