We have previously shown a more potent impact of knockout of Cu,Zn-superoxide dismutase (SOD1) than that of Se-dependent glutathione peroxidase-1 (GPX1) on murine hepatotoxicity induced by an intraperitoneal (ip) injection of a high dose of acetaminophen (APAP, 600 mg/kg). The objective of this experiment was to compare the temporal impacts of knockouts of GPX1 and SOD1 alone or together on mouse susceptibility to an injection of a low dose of APAP (300 mg/kg). The APAP-mediated rises in plasma alanine aminotransferase activity and nitrate/nitrite concentrations, hepatic GSH depletion, and hepatic protein nitration at 5 and (or) 24 h were nearly abolished ( P < 0.05) in SOD1−/− or GPX1 and SOD1 double-knockout (DKO) mice, while GPX1−/− mice exerted only moderate or no change compared with the WT. Despite an increased ( P < 0.05) APAP- N-acetylcysteine and decreased APAP-glucuronide ( P < 0.05) relative to the total APAP metabolites in urine collected for 24 h after the APAP injection, the SOD1−/− mice displayed no shift in urinary APAP-cysteine compared with the WT mice. Knockout of SOD1 prevented the APAP-induced hepatic GPX inactivation ( P < 0.05), whereas knockout of GPX1 aggravated the APAP-induced hepatic SOD activity loss ( P < 0.05). However, the APAP-mediated activity changes of these enzymes in liver accompanied no protein alterations. In conclusion, knockout of GPX1 or SOD1 exerted differential impact on mouse susceptibility to this low dose of APAP, but neither shifted urinary APAP-cysteine formation.