Autophagy is an evolutionarily conserved biological process that degrades intracellular proteins and organelles including damaged mitochondria through the formation of autophagosome. We have previously demonstrated that pharmacological induction of autophagy by rapamycin protects against acetaminophen (APAP)-induced liver injury in mice. In contrast, in the present study, we found that mice with the liver-specific loss of Atg5, an essential autophagy gene, were resistant to APAP-induced liver injury. Hepatocyte-specific deletion of Atg5 resulted in mild liver injury characterized by increased apoptosis and compensatory hepatocyte proliferation. The lack of autophagy in the Atg5-deficient mouse livers was confirmed by increased p62 protein levels and the absence of LC3-lipidation as well as autophagosome formation. Analysis of histological and clinical chemistry parameters indicated that the Atg5 liver-specific knockout mice are resistant to APAP overdose (500 mg/kg). Further investigations revealed that the bioactivation of APAP is normal in Atg5 liver-specific knockout mice although they had lower CYP2E1 expression. There was an increased basal hepatic glutathione (GSH) content and a faster recovery of GSH after APAP treatment due to persistent activation of Nrf2, a transcriptional factor regulating drug detoxification and GSH synthesis gene expression. In addition, we found significantly higher hepatocyte proliferation in the livers of Atg5 liver-specific knockout mice. Taken together, our data suggest that persistent activation of Nrf2 and increased hepatocyte proliferation protect against APAP-induced liver injury in Atg5 liver-specific knockout mice.