Glutamate-mediated cytotoxicity has been implicated in the pathogenesis of neurological diseases, including Parkinson’s disease, Alzheimer’s disease, and stroke. In this study, we investigated the protective effects of alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, on glutamate-induced cytotoxicity in cultured C6 astroglial cells. Exposure to high-dose glutamate (10 mM) caused oxidative stress and mitochondrial dysfunction through the elevation of reactive oxygen species, depletion of glutathione, and loss of the mitochondrial membrane potential (ΔΨ_m). Pretreatment with ALA (200 µM), however, significantly inhibited the glutamate-induced oxidative stress and mitochondrial dysfunction. ALA pretreatment dose-dependently suppressed glutamate-induced apoptotic events including altered nuclear morphology and activation of caspase-3. In addition, ALA significantly attenuated glutamate-induced endoplasmic reticulum (ER) stress markers; namely, glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), protein kinase regulated by RNA (PKR)-like ER-associated kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), inositol-requiring enzyme 1 (IRE1), CCAAT/enhancer binding protein homologous protein (CHOP), and caspase-12. We confirmed that CHOP and caspase-12 are key mediators of glutamate-induced ER stress. Furthermore, exposure of the cells to a caspase-12-specific inhibitor and CHOP small interfering RNAs (siRNAs) led to restoration of the ΔΨ_m that was damaged by glutamate treatment. These results suggest that ALA can effectively suppress oxidative stress, mitochondrial dysfunction, and ER stress in astroglial cells.