An overdose of the most popular analgesic, acetaminophen (APAP), is one of the leading causes of acute liver failure. It is well established that glutathione is exhausted by APAP-reactive intermediate N‑acetyl‑p‑benzoquinone-imine (NAPQI). This leads to elevated phosphorylated-c-Jun N-terminal kinase (p-JNK), which further activates reactive oxygen species (ROS), initiates an inflammatory response, and finally leads to severe hepatic injury. The present study was conducted to investigate the protective role of mangiferin (MAN), a naturally occurring xanthone and anti-oxidant, on APAP-induced hepatotoxicity. C57BL/6 mice were pretreated with or without MAN at 1 h prior to APAP challenge. MAN was administered at a dose of 12.5−50 mg/kg along with APAP at a dose of 400 mg/kg. According to the ALT/AST ratio, 25 mg/kg MAN was the most potent dose for further experiments. Serum ALT and AST depletion were observed in APAP + MAN (25 mg/kg)-treated mice at 6, 12, and 24 h. Early (1 h after APAP treatment) GSH depletion by APAP overdose was restored by MAN treatment, which reduced APAP-Cys adduct formation and promoted protection. p-JNK downregulation and AMPK activation were observed in MAN-treated mice, which could mechanistically reduce oxidative stress and inflammation. MAN up-regulated liver GSH and SOD and reduced lipid peroxidation. HO-1 protein and p47 phox mRNA expression indicated that MAN regulated oxidative stress along with JNK deactivation. The expression of inflammatory response genes TNF-α, IL-6, MCP-1, CXCL-1, and CXCL-2 reached the basal levels after MAN treatment. mRNA, protein, and serum levels of IL-1β were reduced, and NF-κB expression was similar to that of the MAN-treated APAP mice. MAN post-treatment (1 h after APAP treatment) also protected the mice from hepatotoxicity. In conclusion, MAN had a protective and therapeutic role in APAP-induced hepatotoxicity by improving the metabolism of acetaminophen and APAP-Cys adduct formation followed by JNK-mediated oxidative stress and inflammation.