Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, is used to treat hypertension, postpartum hemorrhage, dysentery, and amenor-rhea as a traditional medicine in Asia. We investigated the effect of rutaecarpine on acetaminophen-induced hepatotoxicity in mice. Rutaecarpine was administered orally daily for seven consecutive days, followed by intraperitoneal injection of acetaminophen in mice on day seven to induce hepa-totoxicity. Rutaecarpine pretreatment significantly decreased acetaminophen-induced serum ala-nine aminotransferase (ALT)/aspartate aminotransferase (AST) activities and hepatic malondialde-hyde content and prevented acetaminophen-induced hepatic glutathione depletion. Furthermore, CYP2E1 expression was decreased by rutaecarpine pretreatment in a dose-dependent manner. Ru-taecarpine pretreatment inhibited acetaminophen-induced expression of inflammatory cytokines by inhibiting NF-κB activation by JNK1/2. Also, rutaecarpine pretreatment promoted Nrf2-mediated activation of the antioxidant enzymes GCLC, HO-1, and NQO1. This indicates that the protective effect of rutaecarpine during acetaminophen-induced acute liver injury is mediated by the activation of antioxidant enzymes. Therefore, rutaecarpine has a protective effect of APAP-induced liver damage. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.