Physiopathological conditions such as acute liver failure (ALF) induced by acetaminophen (APAP) can often impair the mitochondrial bioenergetics. Diphenyl diselenide [(PhSe)2] has been shown protects against APAP-induced ALF. The present study aimed to clarify the signaling mechanism involved in the protection of bioenergetics dysfunction associated with ALF-induced by APAP overdose. Mice received APAP (600 mg/kg) or (PhSe)2 (15.6 mg/kg) alone, or APAP +(PhSe)2, all the solutions were administered by the intraperitoneal (i.p.). Samples of liver, blood and liver mitochondria were collected at 2 and 4 h after APAP administration. APAP-induced ALF was able to induce ALF by means of alteration on liver injury biomarkers, increased Nitrite and Nitrate levels and the impairment of oxidative phosphorylation capacity (OXPHOS). In parallel, APAP overdose promoted activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and Heat shock protein 70 (HSP70) expression. (PhSe)2 was able to abolish the APAP-induced decline of OXPHOS and changes on the Nrf2-ARE pathway. In addition, (PhSe)2 elevated the levels of peroxisome proliferator-activated receptor-γ coactivator (PGC-1α), helping to restore the levels of nuclear respiratory factor 1 (NRF1) associated with mitochondrial biogenesis. In summary, the treatment with (PhSe)2 maintained mitochondrial function, promoted genes related to mitochondrial dynamic and demonstrating to play critical role in the modulation of cellular protective responses during ALF.