ABSTRACT Acyl-CoA synthetases (ACSLs) are a family of enzymes that convert intracellular fatty acids into acyl-CoA. A previous study has demonstrated that the yeast ACSL Faa1 (a homolog of mammalian ACSL4) is involved in autophagosome membrane elongation. In the present study, we investigated the involvement of ACSL3, a key enzyme responsible for lipid droplet formation, in autophagosome formation and compared its role with that of ACSL4. Knockdown of ACSL3 impaired starvation-induced autophagy concomitant with the formation of enlarged autophagosome-like structures negative for WIPI2, whereas its overexpression resulted in the formation of WIPI2-positive, but LC3-negative dots, under normal nutrition conditions, likely in an enzymatic activity-independent manner. In contrast, ACSL4 knockdown inhibited starvation-induced autophagosome formation, whereas its overexpression caused autophagosome formation under normal nutrition conditions. Inhibition of autophagosome formation in ACSL4-depleted cells could be rescued by ethanolamine, suggesting a deficit of phosphatidylethanolamine in ACSL4-depleted cells. These results suggest that ACSL3 and ACSL4 are involved in different stages of autophagosome formation – ACSL3 in the formation of fusion-competent autophagosomal membranes and ACSL4 in the formation of autophagosomes.