In our prior research, we documented the robust neuroprotective activity of androst-16-ene-3,6-diol, which was extracted from the methanolic extract of Lysimachia christinae. This compound exhibited significant neuroprotection against glutamate-induced neurotoxicity in HT22 cells, which were subjected to injury caused by an excess of glutamate. In this study, our objective was to validate the neuroprotective effects of androst-16-ene-3,6-diol in HT22 cells subjected to glutamateinduced injury. Additionally, we aimed to elucidate the mechanisms underlying the neuroprotective activity of androst-16-ene-3,6-diol. Androst-16-ene-3,6-diol mitigated the elevated levels of reactive oxygen species induced by excessive glutamate treatment in HT22 cells. Furthermore, the treatment with androst-16-ene-3,6-diol led to a reduction in Ca2+ concentration. The compound also restored the mitochondrial membrane potential to its normal condition. Additionally, androst-16-ene-3,6-diol not only elevated the levels of glutathione reductase and peroxidase to those of the control but also increased the amount of glutathione, an endogenous antioxidant. These results suggested that androst-16-ene-3,6-diol isolated from L. christinae showed potent neuroprotective activity through the anti-oxidative pathway.