Liver injury from paracetamol (acetaminophen) (APAP) is common worldwide. To prevent intoxication with a drug with high poisoning, treatment can be made possible with an easily accessible and harmless substance. This study aimed to investigate the hepa-toprotective effects of Gossypin (GOS) in mice exposed to an overdose of APAP-the possible mechanism of action. Specifically, serum [alanine aminotransferase (ALT), aspartate transaminase (AST), and hepatic biochemical parameters (glutathione (GSH), malondial-dehyde (MDA) and superoxide dismutase (SOD)] were evaluated. Protein and mRNA levels of inflammatory, apoptotic, and cytochrome factors, including TNF-α, IL-1β, IL-6, NF-kB, and CYP2E1, were analyzed using real-time PCR. Pretreatment with GOS signifi-cantly reduced APAP-induced hepatic injury via oxidative stress. Along with potent anti-oxidant activity, GOS promoted APAP hepatic detoxification by regulating AST, ALT, GSH, MDA, and SOD activities and mRNA levels of the cytochrome CYP2E1 gene. The anti-inflammatory activity of GOS increases its production. TNF-α, IL-1β and IL-6, through possible NF-kB blockade, are also responsible for its hepatoprotective effect. Taken together, GOS has the potential to be developed as a preventive agent to be admin-istered to patients suffering from APAP overdose.