In amyotrophic lateral sclerosis, down-regulation of the astrocyte-specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine-antiporter system xc−. Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system xc− and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), a known inducer of system xc−, and the specific xc− subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone-stimulated changes in Nrf2, system xc−, and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell-derived human motor neurons. We conclude that ceftriaxone-mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system xc− than to excitatory amino acid transporter induction.