<p>High-mobility group box 1 (HMGB1) undergoes dynamic expression, release, and subcellular localization changes, and exerts distinct functions in the central nervous system, playing a crucial role in neuroinflammation and exacerbating autoimmune diseases. Although microglia exhibit elevated HMGB1 expression during experimental autoimmune encephalomyelitis (EAE), the precise roles of microglial-derived HMGB1 in the pathogenesis and progression of EAE remain largely unknown. In this study, we generated conditional knockout mice lacking HMGB1 in microglia to assess the role of HMGB1 in EAE progression. We found that depletion of microglial HMGB1 decreased morbidity, delayed the onset of symptoms, and reduced the severity of demyelination in EAE. Furthermore, EAE mice with a conditional knockout of HMGB1 in microglia exhibited decreased expression of CD3⁺ T cells and HMGB1-positive cells in the spinal cord. This resulted in a marked reduction in the number of activated microglia and an alteration in their morphology, thereby restoring the pro-/anti-inflammatory balance of microglia/macrophages; these effects were accompanied by the regulation of inflammatory factor expression and neuronal damage in EAE. Together, these results suggest that HMGB1 derived from microglia breaks the pro-/anti-inflammatory balance and aggravates neuroinflammation in EAE. We propose that targeting microglial HMGB1 could be an effective way to reduce neuroinflammation.</p>