NF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE). In this study, we demonstrate that Nrf2 protects neurons in mixed primary neuronal cultures containing both astrocytes ( approximately 10%) and neurons ( approximately 90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine or rotenone)-induced apoptosis compared with Nrf2+/+ neurons. To understand the underlying mechanism of this observed differential sensitivity, we compared the gene expression profiles using oligonucleotide microarrays. Microarray data showed that Nrf2+/+neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuron-specific proteins, and signaling molecules compared with Nrf2-/- neuronal cultures. As predicted from the microarray data, Nrf2-/- neurons were indeed more vulnerable to the cytotoxic effects of ionomycin- and 2,5-di-(t-butyl)-1,4-hydroquinone-induced increases in intracellular calcium. Finally, adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone- or ionomycin-induced cell death. Taken together, these findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult.