The origin of phagophore membranes for macroautophagy remains unclear. We demonstrate that in mammalian cells and in yeast, starvation induces fusion of mitochondria and the formation of an excess of outer mitochondrial membrane (OMM), which then transforms into multilamellar organelles (MLOs). MLOs acquire SNAP29 and STX17 and are transformed into phagophores that enwrap parts of the cell and form initial autophagic vacuoles (IAVs) and then degradative autophagic vacuoles (DAVs). Membranes of MLO, phagophore, IAV and OMM are similar but distinct from those of the ER, Golgi, PM, endosomes and inner mitochondrial membrane (IMM). During cytokinesis, when fragmented mitochondria fuse, accumulation of MLOs also occurs. Some MLOs fuse with the ER and are transported to the Golgi and lysosomes. In starved neurons, synapses devoid of mitochondria show no signs of autophagy. Thus, phagophores are formed from multilamellar organelles that originate from the outer mitochondrial membrane following mitochondrial fusion.