Ginsenoside Rg 3 (Rg 3 ), one of the active ingredients in Panax ginseng, attenuates NMDA receptor-mediated currents in vitro and antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. In the present study, we examined the neuroprotective effects of Rg 3 on 24-hydroxycholesterol (24-OH-chol)-induced cytotoxicity in vitro. The results showed that Rg 3 treatment significantly and dose-dependently inhibited 24-OH-chol-induced cell death in rat cultured cortical neurons, with an IC 50 value of 28.7 ± 7.5 μm. Furthermore, the Rg 3 treatment not only significantly reduced DNA damage, but also dose-dependently attenuated 24-OH-chol-induced caspase-3 activity. To study the mechanisms underlying the in vitro neuroprotective effects of Rg 3 against 25-OH-chol-induced cytotoxicity, we also examined the effect of Rg 3 on intracellular Ca 2+ elevations in cultured neurons and found that Rg 3 treatment dose-dependently inhibited increases in intracellular Ca 2+, with an IC 50 value of 40.37 ± 12.88 μm. Additionally, Rg 3 treatment dose-dependently inhibited apoptosis with an IC 50 of 47.3 ± 14.2 μm. Finally, after confirming the protective effect of Rg 3 using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, we found that Rg 3 is an active component in ginseng-mediated neuroprotection. These results collectively indicate that Rg 3 -induced neuroprotection against 24-OH-chol in rat cortical neurons might be achieved via inhibition of a 24-OH-chol-mediated Ca 2+ channel. This is the first report to employ cortical neurons to study the neuroprotective effects of Rg 3 against 24-OH-chol. In conclusion, Rg 3 was effective for protecting cells against 24-OH-chol-induced cytotoxicity in rat cortical neurons. This protective ability makes Rg 3 a promising agent in pathologies implicating neurodegeneration such as apoptosis or neuronal cell death.