The individual units (called acini) comprising glandular epithelium possess a hollow lumen that is filled in early epithelial cancers. While investigating the generation of this hollow lumen using an in vitro three-dimensional (3D) epithelial cell culture model, we observed extensive autophagy in dying cells during lumen formation, and thus, proposed that excessive self-eating may promote cell death in the lumen. However, we now reassess this hypothesis. Because cells in the lumen die due to extracellular matrix (ECM) deprivation (anoikis), we tested if autophagy is regulated by ECM detachment. We discovered that detachment strongly induces autophagy in epithelial cells, even when apoptosis is inhibited by Bcl-2 expression. RNAi-mediated depletion of autophagy-related (ATG) genes inhibits detachment-induced autophagy, resulting in increased apoptosis and reduced clonogenic recovery following anoikis. Similarly, during 3D morphogenesis, ATG-depletion enhances luminal apoptosis and fails to elicit long-term luminal survival and filling, even when combined with apoptotic inhibition. Thus, autophagy promotes epithelial cell survival during anoikis. These results broach the possibility that autophagy contributes to the survival of tumor cells lacking appropriate matrix contact, either during early carcinoma formation or in the later stages of dissemination and metastasis.