Nuclear factor-κB (NF-κB) is activated in brain cells after various insults, including cerebral ischemia and epileptic seizures. Although cell culture studies have suggested that the activation of NF-κB can prevent neuronal apoptosis, the role of this transcription factor in neuronal injury in vivo is unclear, and the specific κB subunits involved are unknown. We now report that mice lacking the p50 subunit of NF-κB exhibit increased damage to hippocampal pyramidal neurons after administration of the excitotoxin kainate. Gel-shift analyses showed that p50 is required for the majority of κB DNA-binding activity in hippocampus. Intraventricular administration of κB decoy DNA before kainate administration in wild-type mice resulted in an enhancement of damage to hippocampal pyramidal neurons, indicating that reduced NF-κB activity was sufficient to account for the enhanced excitotoxic neuronal injury in p50^−/− mice. Cultured hippocampal neurons from p50^−/− mice exhibited enhanced elevations of intracellular calcium levels and increased levels of oxidative stress after exposure to glutamate and were more vulnerable to excitotoxicity than were neurons from p50^+/+ and p50^+/− mice. Collectively, our data demonstrate an important role for the p50 subunit of NF-κB in protecting neurons against excitotoxic cell death.