The selective removal of undesired or damaged mitochondria by autophagy， known as mitophagy， is crucial for cellular homoeostasis， and prevents tumour diffusion， neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here， we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy， a process that takes place independently of the main mitophagy receptors. Furthermore， we show that mitophagy function of AMBRA1 is post-translationally controlled， upon HUWE1 activity， by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1， thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether， these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway， in which HUWE1 and IKKα are key factors， shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.