Free Zn2+ is released in excess at excitatory synapses in pathological conditions including transient global and focal cerebral ischemia, which causes neuronal and glial cell death. In the current study, we explored the mechanism underlying Zn2+-induced cell death in primary cortical astroglial cultures. Chronic treatment with 30–35 µM Zn2+ led to the death of 70–95% of astrocytes within 18 h, preceded by Zn2+ influx. Extracellular glutathione (GSH; 100 µM) completely blocked the Zn2+ influx and Zn2+ toxicity. The Zn2+ toxicity was also inhibited when intracellular GSH was increased. Conversely, it was aggravated when intracellular GSH was depleted by buthionine sulfoximine (BSO). Consistently, the level of cellular GSH was notably decreased with a concurrent increase in oxidized GSH in Zn2+-treated astrocytes. These results suggest that the disruption of proper maintenance of thiol homeostasis is a mechanism underlying Zn2+ toxicity in primary cortical astrocytes.