Acetaminophen (APAP) is one of the most commonly used oral analgesics and antipyretics, but hepatotoxicity including liver failure may occur after overdose. The therapeutic options for treating APAP hepatotoxicity are limited. Eriodictyol, a dietary flavonoid with anti-inflammatory and antioxidant properties, was used here to determine its protective effects against APAP-induced hepatotoxicity in mice. Various administration routes and pharmacokinetics-pharmacodynamics (PK-PD) analyses were used to determine these effects. Protective effects were observed in intravenously and intraperitoneally but not in intragastrically administered eriodictyol. LC-MS/MS analysis revealed two monoglucuronide metabolites of eriodictyol in liver and intestine microsomes. Recombinant human uridine-5'-diphospho -glucuronosyltransferase (UGT) isoforms and chemical inhibition studies demonstrated that UGT1As (mainly UGT1A1, UGT1A9, UGT1A10) and UGT2B7 were likely the main contributors to eriodictyol glucuronidation. Intragastric administration of eriodictyol, which displayed lower parent and higher metabolite concentrations in the plasma, did not elicit protective effects against APAP hepatotoxicity, when compared to the intraperitoneal injection of eriodictyol. The relative bioavailability of eriodictyol was increased to 216.84% with the coadministration of glycyrrhetinic acid (GA), an inhibitor of UGT1As. Intragastric administration of eriodictyol in combination with GA also induced protective effects against APAP hepatotoxicity. Furthermore, intragastric administration of eriodictyol attenuated APAP hepatotoxicity in heterozygous Ugt1 (Ugt1) mice but not in its wild-type littermates. Thus, UGT1A-mediated metabolic inactivation reduced the protective effect of eriodictyol. Eriodictyol attenuated APAP hepatotoxicity via inhibition of hepatic cytochrome P450 (cyp) 2e1 and cyp3a11 activities; reserve of glutathione (GSH) by improvement of glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST) activities; elevation of superoxide dismutase (SOD) activity; and reduction of malondialdehyde (MDA) level. Our findings indicate that parenterally administered eriodictyol may be used to treat APAP-induced hepatotoxicity, and its efficacy can be enhanced by UGT1As down-regulation.