Mitochondria control both cell’s life and death. To ensure the well-being of the cell, mitochondrial quality must be tightly monitored through a mechanism of mitochondrial autophagy or mitophagy, which selectively removes damaged or unwanted mitochondria. We recently have identified that mitochondrial outer membrane protein FUNDC1 harbors LC3-interacting region (LIR) and interacts with LC3 to mediate mitophagy upon hypoxic treatment. Under normoxic condition, FUNDC1 is highly phosphorylated by Src kinase and CK2 at Try18 and Ser13, respectively. Mitochondrial stresses and hypoxic treatment lead to the dephosphorylation of FUNDC1 that has higher affinity with LC3. Importantly, we have identified that mitochondrially localized phosphatase PGAM5 is able to dephosphorylate FUNDC1 at Ser-13. The phosphatase activities of PGAM5 is inhibited through its interaction with Bcl-xL, and the degradation of Bcl-xL could lead to the activation of PGAM5 for mitophagy. Our results delineate the signaling pathway linking mitochondrial stresses and hypoxia towards the activation of mitophagy.