Leonurine (Leo) is a natural alkaloid isolated from the herb Leonurus japonicus Houtt. (Leonuri) that has been shown to inhibit oxidative stress and inflammation. However, the role and mechanism of Leo in acetaminophen (APAP)-induced acute liver injury (ALI) remain unknown. In this study, we investigated the protective effect of Leo against APAP-induced ALI and elucidated the molecular mechanism. Here, we showed that the damage to mouse primary hepatocytes (MPHs) induced by APAP was attenuated by treatment with Leo, which promoted proliferation and inhibited oxidative stress injury, and Leo significantly improved APAP-induced ALI in mice. Leo could protect against APAP-induced ALI by reducing serum aspartate aminotransferase (AST) and alanine transaminase (ALT) levels, hepatic histopathological damage, liver cell necrosis, inflammation, and oxidative stress-induced damage in vivo and in vitro. Moreover, the results indicated that Leo relieved APAP-induced liver cell necrosis by reducing the expression of Bax and cleaved caspase-3 and increasing Bcl-2 expression. Leo alleviated APAP-induced oxidative stress-induced damage by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, which facilitated Nrf2 nuclear translocation and upregulated oxidative stress-related protein expression in liver tissues. Moreover, the results suggested that APAP-induced inflammation in the liver was suppressed by Leo by suppressing the Toll-like receptor 4 (TLR4) and NLR family pyrin domain containing 3 (NLRP3) pathways. In addition, Leo facilitated the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in the liver tissue of ALI mice. Network pharmacology, molecular docking, and western blotting showed that PI3K was a potential target of Leo in the treatment of ALI. Molecular docking and cellular thermal shift assay (CETSA) indicated that Leo could stably bind to the PI3K protein. In conclusion, Leo attenuated ALI, and reversed liver cell necrosis, the inflammatory response and oxidative stress-induced damage by regulating the PI3K/AKT signaling pathway. CI - Copyright (c) 2023 The Authors. Published by Elsevier B.V. All rights reserved.