The critical importance of glutathione in mitigating the deleterious effects of electrophile generating drugs such as acetaminophen (APAP) is well established. However, the role of other antioxidant systems, such as that provided by thioredoxin, has not been extensively studied. Selenoprotein thioredoxin reductase 1 (Txnrd1) is important for attenuating activation of the apoptosis signaling-regulating kinase 1 (ASK1) and the c-Jun N-terminal kinase (JNK) pathway caused by high doses of APAP. Therefore, a detailed investigation of the role of Txnrd1 in APAP-induced hepatotoxicity was conducted. Liver-specific Txnrd1 knockout mice (Txnrd1ΔLiv) were generated and treated with a hepatotoxic dose (400 mg/kg) of APAP for 1 or 6 h. Liver toxicity was assessed by measuring the activities of liver enzymes aspartate aminotransferase and alanine aminotransferase in serum, in addition to histopathological analysis of liver sections and analysis of glutathione levels. At 1 h post-APAP treatment, total and mitochondrial glutathione levels in control and Txnrd1ΔLiv mice were similarly depleted. However, at 6 h post-APAP treatment, Txnrd1ΔLiv mice were resistant to APAP toxicity as liver enzymes and histology were not significantly different from the corresponding untreated mice. Analyses revealed the compensatory up-regulation of many of the nuclear factor erythroid 2-related factor 2 (NRF2) target genes and proteins in Txnrd1ΔLiv mice with and without APAP treatment. Yet, JNK was phosphorylated to a similar extent in APAP-treated control mice. The results suggest that Txnrd1ΔLiv mice are primed for xenobiotic detoxication primarily through NRF2 activation.