Acetaminophen (APAP) overdose is the most frequent cause of adult acute liver failure. Susceptibility or resistance to APAP toxicity is most likely accounted for by the interplay of several factors. One factor important in multiple different chronic liver diseases that may play a role in APAP toxicity is elevated hepatic iron. Hereditary hemochromatosis is traditionally associated with hepatic iron overload. However, varying degrees of elevated hepatic iron stores observed in chronic hepatitis C and B, alcoholic liver disease and nonalcoholic fatty liver disease also have clinical relevance. We employed an animal model in which mice are fed a 3,5,5-trimethyl-hexanoyl-ferrocene (TMHF)-supplemented diet to evaluate the effect of elevated hepatic iron on APAP hepatotoxicity. Three hundred milligrams per kilogram APAP was chosen because this dosage induces hepatotoxicity but is not lethal. Since both excess iron and APAP induce oxidative stress and mitochondrial dysfunction, we hypothesized that the TMHF diet would enhance APAP hepatotoxicity. The results were the opposite. Centrilobular vacuolation/necrosis, APAP adducts, nitrotyrosine adducts, and a spike in serum alanine aminotransferase, which were observed in control mice treated with APAP, were not observed in TMHF-fed mice treated with APAP. Further analysis showed that the levels of CYP2E1 and CYP1A2 were not significantly different in TMHF-treated compared with control mice. However, the magnitude of depletion of glutathione following APAP treatment was considerably less in TMHF-treated mice than in mice fed a control diet. We conclude that a TMHF diet protects mice from moderate transient APAP-induced hepatotoxicity prior to the formation of APAP adducts, and one contributing mechanism is reduction in glutathione depletion.