AbstractExcitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N‐methyl‐d‐aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology‐specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24‐h post‐insult. Egr‐1 and NOR‐1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA‐induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up‐regulated pathogenic genes, down‐regulated pro‐survival genes) and opposing compensatory responses (down‐regulated pathogenic genes, up‐regulated pro‐survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF‐κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF‐κB inhibitor MG‐132 indeed altered NMDA‐mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi‐target treatment strategies and implications for predictive medicine.