Heat stroke (HS) is a potentially fatal acute condition caused by an interplay of complex events including inflammation, endothelial injury, and coagulation abnormalities that make its pharmacological treatment a challenging problem. The traditional Chinese medicine Xuebijing injection (XBJ) has been shown to reduce inflammatory responses and prevent organ injuries in HS-induced mice. However, the underlying mechanism of XBJ in HS-induced brain injury remains unclear. In this study, HS-induced rat models and cell models were established to elucidate the effects and underlying mechanisms of XBJ injection on HS-induced brain injury in vivo and in vitro. The results revealed that XBJ injection improved the survival outcome of HS rats and attenuated HS-induced brain injury in a concentration-dependent manner. Subsequently, the reduction in viability and proliferation of neurons induced by HS were reversed by XBJ treatment, while the HS-induced increased ROS levels and neuron death were also inhibited by XBJ injection. Mechanistically, HS activated PARP-1/AIF signaling in vitro and in vivo, inducing the translocation of AIF from the cytoplasm to the nucleus, leading to PARP-1-dependent cell death of neurons. Additionally, we compared XBJ injection effects in young and old age rats. Results showed that XBJ also provided protective effects in HS-induced brain injury in aging rats; however, the treatment efficacy of XBJ injection at the same concentration was more significant in the young age rats. In conclusion, XBJ injection attenuates HS-induced brain injury by inhibiting oxidative stress and Parthanatos via the PARP-1/AIF signaling, which might provide a novel therapeutic strategy for HS treatment.© 2023 The Authors. Published by American Chemical Society.