Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double-membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long-standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant-negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)-II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.