Abstract: [Objective] To investigate the inhibitory effect of Yinchen Shensha Hewei Powder on hyperbilirubinemia-induced brain injury in neonatal rats and its regulatory mechanism on the HMGB1/TLRs/NF-κB signaling pathway, so as to provide experimental evidence for clinical application. [Methods] Sixty 7-day-old Sprague-Dawley neonatal rats were randomly divided into a model control group, a positive control group, a Yinchen Shensha Hewei Powder group, a Yinchen Shensha Hewei Powder + HMGB1 inhibitor group, a Yinchen Shensha Hewei Powder + TLRs inhibitor group, and a Yinchen Shensha Hewei Powder + NF-κB inhibitor group, with 10 rats in each group. Rat models of hyperbilirubinemia-induced brain injury were established by intraperitoneal injection of bilirubin. After modeling, each experimental group was administered the corresponding intervention for 7 consecutive days. Neurological function was evaluated using the Tarlov score. Serum and brain bilirubin levels were detected by colorimetry. Pathological changes in brain tissue were observed by hematoxylin-eosin staining. The positive expressions of NF-κB, NSE and NF proteins in brain tissue were detected by immunohistochemistry. The mRNA expression levels of HMGB1, TLR4, NF-κB, COX-2 and iNOS in brain tissue were measured by quantitative real-time polymerase chain reaction. Statistical analysis was performed using SPSS 24.0 software with one-way analysis of variance for comparison of quantitative data among multiple groups. A P value < 0.05 indicated a statistically significant difference. This study was approved by the Laboratory Animal Management and Ethics Committee of Wenzhou Medical University (approval No. xms92022-0677). [Results] Compared with the model control group, the positive control group and the Yinchen Shensha Hewei Powder group showed significantly increased Tarlov scores (P<0.05), markedly decreased serum and brain bilirubin levels (P<0.01), obviously alleviated pathological injury in brain tissue, and significantly downregulated expressions of inflammation-related proteins and mRNAs (P<0.01). The combined inhibitor groups exhibited no significant difference or slight improvement in the above indicators compared with the traditional Chinese medicine alone group, showing a synergistic regulatory trend. Yinchen Shensha Hewei Powder significantly attenuated hyperbilirubin-induced brain injury, neuronal apoptosis and inflammatory cell infiltration. Its protective effect was related to the inhibition of excessive activation of the HMGB1/TLRs/NF-κB signaling pathway . [Conclusion] Yinchen Shensha Hewei Powder effectively alleviates hyperbilirubinemia-induced brain injury in neonatal rats. Its mechanism is mainly related to inhibiting abnormal activation of the HMGB1/TLRs/NF-κB inflammatory signaling pathway, downregulating the expression of inflammatory factors and reducing the release of proinflammatory mediators. This study provides experimental evidence for the clinical application of Yinchen Shensha Hewei Powder in the treatment of neonatal hyperbilirubinemia-associated brain injury and lays a foundation for optimizing dosage regimens and developing precise clinical protocols. Large-sample clinical studies can be further conducted to verify its clinical efficacy and safety in humans.