OBJECTIVES: Improper usage of acetaminophen (APAP) leads to morbidity and also mortality secondary to liver damage. Ginseng could suppress APAP-induced hepatotoxicity and ginsenoside Rg3 is a kind of major component in ginseng against liver damage. Herein, we intended to estimate the beneficial function and molecular mechanism of Rg3 on APAP-caused hepatotoxicity and identified hepatoprotection. METHODS: A total of 50 C57BL/6J mice were divided into five random groups, and each contains 10 mice as the control, acetaminophen (350 mg/kg) and Rg3 (5, 10 and 20 mg/kg) + acetaminophen (350 mg/kg) groups. These mice were intragastric administration a single dose of acetaminophen by oral treatment behind pre-administered with several doses of ginsenoside Rg3 for six hours. KEY FINDINGS: According to our data, the injection of APAP (350 mg/kg) enhanced the basal levels of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and lactic dehydrogenase. However, these abnormal added were alleviated by Rg3. Moreover, Rg3 treatment obviously relieved APAP-caused inflammation and oxidant in liver tissues. The depletion of glutathione, glutathione peroxidase, total antioxidant capacity and generation of malondialdehyde induced by APAP treatment were reduced by Rg3. By H&E staining, Rg3 effectively reduced APAP-caused apoptosis and inflammatory infiltration. Moreover, Rg3 attenuated APAP-caused hepatic damage in part by regulating the pro-inflammatory and anti-inflammatory cytokines. Moreover, we found that Rg3 could bind to NLRP3 suggesting the anti-inflammatory effects of Rg3 by molecular docking study. CONCLUSIONS: In summary, Rg3 showed hepatic protective function in APAP-induced hepatotoxicity as evidenced by a reduction of the oxidant and the inflammatory reply, relieve of hepatocellular damage, showing potential in Rg3 as a potential therapeutic medicine to prevent hepatic injury. © The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: [email protected]