Cepharanthine (CEP) is an alkaloid extracted from Stephania cephalantha Hayata, a traditional Chinese medicine (TCM) renowned for its heatclearing and dehumidifying properties. For centuries, Stephania cephalantha Hayata has been employed in the treatment of a wide range of diseases, including pain, edema, inflammation, and fever.Our research aims to investigate the role and mechanism of Cepharanthine in ameliorating uric acid (UA) induced neuroinflammatory responses.The Connectivity Map (CMap) was utilized to identify the therapeutic drug Cepharanthine, based on the proteomic disturbances associated with uric acid (UA). Limited proteolysis small molecule mapping (LiP-SMap) and thermal proteome profiling (TPP) technologies were used to identify the direct target proteins for UA and Cepharanthine. Additionally, we used the induced-fit docking algorithm integrated within the Schrodinger suite to explore the interactions between Cepharanthine and uric acid targets. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology was employed to determine the concentration of Cepharanthine in the mice cerebral cortex. The pro-inflammatory cytokine genes were also quantified by qPCR in U251 cells and in hyperuricemic mice.The findings indicated that uric acid increased the transcription of pro-inflammatory cytokines and the expression levels of proteins linked to inflammation in U251 cells. PPP2R1A was identified as a potential candidate for direct interaction with uric acid, potentially initiating inflammation. Based on the CMap prediction, Cepharanthine was identified as a candidate drug for interaction with PPP2R1A. TPP analysis indicated that Cepharanthine could reduce the thermal stability of PPP2R1A. Molecular docking confirmed that Cepharanthine could directly bind to PPP2R1A. Furthermore, the detection of Cepharanthine in the cerebral cortex suggested its ability to cross the blood-brain barrier. Proteomic analysis of Cepharanthine-treated mice revealed significant enrichments of differentially expressed proteins (DEPs) in inflammation-related pathways and biological processes. Additionally, Cepharanthine was effective in decreasing the expression of pro-inflammatory cytokine genes induced by uric acid in U251 cells and in hyperuricemic mice.Cepharanthine could effectively alleviate hyperuricemia-induced neuroinflammation via binding to PPP2R1A. This study offers a novel approach for prevention and treatment of neurological diseases caused by hyperuricemia.Copyright © 2025. Published by Elsevier B.V.