Abstract: Objective: To analyze the key target and related signal pathway mechanism of antipyretic of Guizhi, one of the “ten herbs of Guangxi” by bioinformatics, and verify them by animal experiments.Methods: TCMSP was used to screen the main active components of Guizhi, and PharmMapper reverse docking was used to predict the target of the active components.The febrile related targets were predicted by the Gene Cards, CTD and OMIM databases.The common targets of Guizhi and febrile were obtained by intersection mapping, and the regulatory network and protein interaction network were established using Cytoscape 3.8.0 software.GO biological function annotation and KEGG pathway enrichment analysis were performed on intersection targets.Finally, the results of network pharmacology enrichment analysis were verified by the whole animal, biochemical and ELISA assays.Results: Eight active components of Guizhi and their 228 predicted targets as well as 12, 882 fever-related targets were screened out.There were 213 common genes between the targets of Guizhi and febrile, including ALB, AKT1, SRC, EGFR, HSP90AA1, etc, which were enriched into the arachidonic acid metabolic signaling pathway, PPAR signaling pathway and NF-κB signal pathway, etc, as was indicated by the KEGG analysis.The results of confirmatory experiments showed that Guizhi could alleviate febrile rats induced by yeast for 6-8 h, which was evidenced by a significant decrease in the changes of body temperature (△T), the maximum net increment of febrile (△Tmax) and the body temperature response index (TRI_8h) (all P＜ 0.05).Guizhi administration significantly decreased the yeast-induced high levels of the peripheral endogenous pyrogen and inflammatory factors, including IL-1β, TNF-α, IL-6, PGE₂ in serum of rats in a dose-dependent manner (P＜ 0.01).High dose could significantly reduce the abnormal levels of central endogenous thermogenic factors such as IL-1β and PGE₂ in the brain tissue of febrile rats (P＜ 0.01), while medium and low dose of Guizhi could only significantly reduce the abnormal levels of IL-1β (all P＜ 0.05), and did not exert a significant effect on PGE₂ (P＞ 0.05); the high and medium doses of Guizhi could significantly decrease COX-1, COX-2 and PGDH1/15-PGDH activities in the brain tissue of febrile rats (all P＜ 0.05), while the low dose of Guizhi could significantly reduce COX-1 and COX-2 activities (all P＜ 0.05), and had no significant effect on abnormally increased PGDH1/15-PGDH activities (P＞ 0.05).Conclusion: Guizhi has a certain antipyretic effect on yeast-induced febrile; its mechanism is related to the weakened activity of key enzymes such as COX-1, COX-2, PGDH1/15-PGDH in the arachidonic acid-pGE₂ synthesis and decomposition pathway, and the reduction of the levels of IL-1β, TNF-α, IL-6 and PGE₂ in central and peripheral areas.