Whether the autophagosomal membrane is derived from the endoplasmic reticulum (ER), mitochondria or the plasma membrane has been controversial. Here, Hamasaki et al. find that, in mammalian cells, autophagosomes originate at ER–mitochondrion contact sites. During starvation the pre-autophagosome marker ATG14 accumulated at ER–mitochondrion contact sites, and, similarly, co-fractionated with mitochondrion-associated ER membranes (which contain the contact sites). Moreover, ATG5, which is involved in autophagosome elongation and closure, also localized at ER–mitochondrion contact sites. Interestingly, knockdown of the ER SNARE protein STX17 during starvation hindered the localization of ATG14 specifically at these contact sites and decreased autophagy, indicating a crucial role for STX17-dependent ATG14 recruitment in autophagosome formation. The finding that ER–mitochondrion contact sites act as autophagosome membrane donors explains why previous studies had obtained evidence for both the ER and mitochondria in this process.