As a traditional Chinese medicine, raw Moutan Cortex (RMC) has been used in clinical practice for thousands of years. However, its blood-cooling and blood-activating medicinal effects as well as the underlying mechanisms have not been preliminarily verified until recent years.Our group's previous network pharmacological studies suggested that RMC might exert its blood-activating and anti-inflammatory effects by modulating the coagulation-inflammation cascade pathway. Therefore, the present study aimed to further investigate the mechanisms relevant to the blood-activating and anti-inflammatory effects of RMC so as to provide more robust data supporting its clinical application.The inflammation and coagulation models of human umbilical vein endothelial cells (HUVECs) were induced by TNF-α; The rat models with blood-heat and blood-stasis syndrome (BHS) were constructed by ice-water bath with a combined use of epinephrine hydrochloride and dried yeast; The thrombus models of zebrafish were induced by arachidonic acid, and the inflammation models were established using LPS and CuSO4. The regulatory effects of RMC on the key targets in the pathway of the coagulation-inflammation cascade were investigated by combining ELISA, RT-PCR, and western blot techniques in an attempt to provide multiple validations concerning RMC's pharmacological efficacy and mechanism associated with cooling blood and activating blood circulation.The findings from the pharmacodynamic research demonstrated that RMC could inhibit the coagulation and inflammation process of HUVECs. Besides, it lowered the anal temperature and whole blood viscosity in BHS rats in addition to a prolongation of their prothrombin time (PT), thrombin time (TT), and activated partial thromboplastin time (APTT). Successfully constrained thrombotic area and reduced inflammatory cell counts were also observed in zebrafish models. Meanwhile, ELISA, RT-PCR and WB showed that RMC were capable of inhibiting the factors related to coagulation-MARK inflammation pathway-FⅡ, TF, FⅦ, FⅧ, FⅩ, and PAI, as well as down-regulating the expression of IL-6, COX-2, iNOS, TNF-α, ERK, JNK and p38.RMC exerts blood-activating and anti-inflammatory effects through regulating the target genes of the coagulation-MARK inflammation cascade pathway.Copyright © 2023. Published by Elsevier B.V.