The endoplasmic reticulum (ER) is a membranous network within cells that is important for several cellular functions including translation and folding of secretory and membrane proteins, lipid biogenesis and sequestration of Ca2+. Disruption of ER structure might affect the normal physiology of the cells. In yeast, expansion of the ER is observed under unfolded protein response (UPR) and subsequently induces autophagy initiated from the ER. In this study, we demonstrated a drastic and specific ER membrane reorganization (EMR), characterized by the clustering of the ER membrane into large and compact aggregates and occurring independent of UPR in HeLa cells treated with S1 combined with ABT-737. Subsequently, combined with S1 and ABT-737 triggered autophagy. Herein, we report a key step for removal of damaged and superfluous cellular constituents, by a mechanistic link between ER aggregation and autophagic activation. Our study is the first time to show that autophagy may be a way to remove the ER membrane reorganization induced by Bcl-2 inhibitors ABT-737 and S1 and it may help us to analyze autophagy in certain diseases.