Macroautophagy is an intracellular degradation process that requires multiple autophagy-related () genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified as a novel gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in -knockout (KO) cells. The phenotype of -KO cells resembled those of -KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in -KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.