We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.