Astrocytes have emerged as critical elements for the maintenance and function of the central nervous system. The expression on their cell membrane of RAGE and TLR4 receptors makes astrocytes susceptible to High-mobility group box 1 (HMGB1), a nuclear protein typically released in the extracellular milieu by living cells experiencing physiological stress conditions or by damaged cells. Here, we show that the interaction of HMGB1 with normal spinal cord astrocytes induces the astrocytic production of neurotrophic factors, particularly brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Multiple investigations suggest a role for HMGB1 in amyotrophic lateral sclerosis (ALS). Yet, no mechanistic information on the implication of HMGB1 signaling in this disorder is currently available. We demonstrate that non-transgenic and transgenic SOD1WT spinal motor neurons exhibit only a basal nucleus-to-cytoplasm shuttling of the HMGB1 protein. Conversely, in SOD1G93A ALS mouse spinal cords, HMGB1 significantly translocates from the nucleus to the cytoplasm of motor neurons, thereby suggesting that it may be eventually released in the extracellular environment during the progression of the disease. We postulate that extracellular HMGB1 can paracrinally interact with the neighboring astrocytes in an attempt to counteract the neurodegenerative process. Yet, at variance with normal cells, SOD1G93A-expressing astrocytes show impaired capacity to raise BDNF and GDNF levels upon HMGB1 stimulation. Our data suggest that HMGB1 have a potential to promote neuroprotective actions by healthy astrocytes. However, this neurotrophic response is disrupted in ALS astrocytes. This indicates that diseased astroglial cells may exacerbate motor neuron degeneration in ALS because of the loss of their neurosupportive functions.