Heat stroke (HS) is a life-threatening condition with complex underlying mechanisms, posing challenges for pharmacological treatment. Xuebijing (XBJ) can effectively relieve HS-induced brain injury, but its molecular mechanism is not well-established. This study was conducted to investigate the mechanisms underlying the protective role of XBJ in HS-induced brain injury. HS-induced mice and cell models were established to elucidate the protective effects and underlying mechanisms of XBJ on HS-induced brain injury in vivo and in vitro. HMGB1 knockout (HMGB1-/-) mice and HMGB1 silencing in primary neuronal cells were used to study the effects of XBJ on HMGB1 in HS. Assessments included survival rate, neuronal damage score, and pathological changes. Various techniques such as Western blot, Transmission Electron Microscope (TEM), immunofluorescence staining, RT-qPCR, commercial kits, TUNEL assay, CCK-8, EdU, flow cytometry, and Co-IP assay were employed to assess autophagy, reactive oxygen species (ROS) levels, oxidative stress, inflammation, neuronal apoptosis, and protein complexes. Data revealed that XBJ ameliorated brain damage and neuron apoptosis in HS-exposed mice and promoted autophagy while inhibiting oxidative stress and inflammatory responses, both in vivo and in vitro. Additionally, XBJ alleviated neuronal brain damage, neuron apoptosis, oxidative stress, and inflammatory responses in HS via inducing autophagy. Furthermore, XBJ promoted the cytoplasmic translocation of HMGB1 from the nucleus and competed with Bcl-2 for binding to Beclin1. Moreover, HMGB1-/- mice and HMGB1 silencing in primary neuronal cells displayed reduced autophagy and enhanced inflammatory responses, both in vivo and in vitro. XBJ protects against HS-induced brain injury via a mechanism involving the autophagy-inflammation pathway mediated by HMGB1.