Most transmembrane proteins are conventionally transported through the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane. Recently, some integral membrane proteins have been found to reach the plasma membrane via unconventional protein secretion (UPS) pathways that bypass the Golgi apparatus under certain conditions. However, mechanisms for this UPS pathways are still not fully delineated. Here, we report that components of autophagic and ESCRT pathways are involved in unconventional secretion of the transmembrane protein CFTR. In HEK293 and HeLa cells, induction of ER stress by thapsigargin or blockade of ER-to-Golgi transport evoked activation of autophagic pathways. Immunoelectron microscopy indicated that GRASP55, which is known to mediate unconventional trafficking of CFTR, is present in the autophagosome and multivesicular body (MVB) under these conditions. A custom small-interfering RNA screen of mammalian ESCRTs revealed that single silencing of some ESCRT components including MVB12B inhibited the unconventional secretion of ΔF508-CFTR. Taken together, these results suggest that components involved in early autophagosome formation and the following MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.