To explore the novel protective effect of Asperosaponin VI (AVI) on sepsis and its potential mechanism.In in vitro experiments, bone marrow mononuclear cells and THP-1-derived cells were used to evaluate the viability of AVI treatment. Besides, the quantitative real-time PCR and Western blot were adopted to explore the protective effect of AVI on LPS-induced inflammation. For in vivo work, the effect of AVI on mice was evaluated by using both CLP-induced and the LPS-induced sepsis mice model. The fluctuation of anal temperature and the behavior of mice were recorded after surgery. Further, the content of bacteria in peritoneal lavage fluid was detected, as well as the levels of ALT, AST, LD and LDH in serum with ELISA. H&E staining and real-time PCR were used to evaluate the histopathology of liver, spleen and lung. Finally, relevant signaling pathways were detected by Western blot, real-time PCR and immunohistochemistry.AVI inhibited the expression of inflammatory factors in both CLP-induced and LPS-induced sepsis mice models, and reduced the number of bacteria in abdominal lavage fluid. The preventive treatment with AVI alleviated sepsis-induced organ injuries, reduced inflammatory responses, which was through inhibiting Hippo and Rho signaling pathway.This study indicated that AVI effectively protected mice from sepsis by down-regulating the activation of Hippo signaling and Rho family, and reducing inflammation and organ damage. However, conventional treatment was using antibiotics, and its mechanism was different with AVI.Copyright © 2022 Elsevier GmbH. All rights reserved.