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 hepatoprotective ef-fects 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), malondialdehyde (MDA) and super-oxide dismutase (SOD)] were evaluated. Protein and mRNA levels of inflammatory, apoptot-ic, and cytochrome factors, including TNF-α, IL-1β, IL-6, NF-kB, and CYP2E1, were ana-lyzed using real-time PCR. Pretreatment with GOS significantly reduced APAP-induced he-patic injury via oxidative stress. Along with potent antioxidant 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 in-creases 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 devel-oped as a preventive agent to be administered to patients suffering from APAP overdose.