Acetaminophen (AA) is a widely used antipyretic drug that causes hepatotoxicity at high doses. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that mitigates electrophilic stress from AA by inducing genes, such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), multidrug resistance-associated proteins (Mrps), and glutathione (GSH) synthesis enzymes. To determine whether Nrf2 activation alters the biotransformation and excretion of AA, male wild-type, Nrf2-null, and Keap1 (Kelch-like ECH-associated protein 1)-knockdown (Keap1-kd) mice (which have increased activation of Nrf2) were administered a single subtoxic dose of AA (50 mg/kg, iv), after which, AA and its metabolites (AA-glucuronide [AA-GLUC]; AA-sulfate [AA-SULF]; AA-glutathione [AA-GSH]) were quantified in plasma, bile, and liver. AA-GLUC concentrations were reduced in plasma and elevated in livers of Nrf2-null mice due to decreased glucuronidation activity and lower expression of the basolateral efflux transporter Mrp3. In contrast, Keap1-kd mice had higher plasma and lower hepatic AA-GLUC concentrations, due to higher Mrp3 expression. Lower glucuronidation activity of Nrf2-null mice increased the proportion of AA available for sulfation, resulting in elevated AA-SULF concentrations in plasma, bile, and liver. Decreased AA-sulfation activity in Keap1-kd mice resulted in lower AA-SULF concentrations. AA-GSH conjugates were increased in Nrf2-null mice and tended to be lower in Keap1-kd mice. Furthermore, Nqo1, an enzyme capable of detoxifying the reactive intermediate of AA metabolism, N-acetyl-p-benzoquinone imine (NAPQI), had 85% lower activity in Nrf2-null mice and 415% higher activity in Keap1-kd mice relative to wild-type. In conclusion, lack of Nrf2 results in decreased AA glucuronidation, leading to increased AA available for NAPQI formation and decreased efflux of AA-GLUC via Mrp3; however, activation of Nrf2, as in Keap1-kd mice, results in decreased sulfotransferase activity, decreased AA-SULF formation, and enhanced elimination of AA-GLUC due to increased expression of Mrp3.