A long-standing quest in the autophagy field is to define the membrane origin of the autophagosome. We have established a cell-free assay based on LC3 lipidation that recapitulates multiple regulatory hallmarks of early autophagosome biogenesis. Using a systematic membrane fractionation approach, we have identified the ER-Golgi intermediate compartment (ERGIC) as the most efficient membrane substrate for LC3 lipidation. Further studies indicate that the ERGIC plays an essential role to trigger LC3 lipidation and autophagosome biogenesis by recruiting the key early autophagic factor ATG14.