High mobility group proteins are chromatin binding factors with key roles in maintenance of nuclear homeostasis. The evidence indicates that extracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promoting inflammation and participating to the pathogenesis of several disorders in peripheral organs. In this study, we have investigated the expression levels and relocation dynamics of HMGB1 in neural cells, as well as its neuropathological potential. We report that HMGB1 is released in the culture media of neurons and astrocytes challenged with necrotic but not apoptotic stimuli. Recombinant HMGB1 prompts induction of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2, interleukin-1β, and tumor necrosis factor α, and increases excitotoxic as well as ischemic neuronal death in vitro. Dexamethasone reduces HMGB1 dependent immune glia activation, having no effect on the protein’s neurotoxic effects. HMGB1 is expressed in the nucleus of neurons and astrocytes of the mouse brain, and promptly (1 h) translocates into the cytoplasm of neurons within the ischemic brain. Brain microinjection of HMGB1 increases the transcript levels of pro-inflammatory mediators and sensitizes the tissue to the ischemic injury. Together, data underscore the neuropathological role of nuclear HMGB1, and point to the protein as a mediator of post-ischemic brain damage.