Sphingolipids are structural lipid components of cell membranes, including membrane of organelles, such as mitochondria or endoplasmic reticulum, playing a role in signal transduction as well as in the transport and intermixing of cell membranes. Sphingolipid microdomains, also called lipid rafts, participate in several metabolic and catabolic cell processes, including apoptosis. However, the defined role of lipid rafts in the autophagic flux is still unknown. In the present study we analyzed the role of gangliosides, a class of sphingolipids, in autolysosome morphogenesis in human and murine primary fibroblasts by means of biochemical and analytical cytology methods. Upon induction of autophagy, by using amino acid deprivation as well as tunicamycin, we found that GD3 ganglioside, considered as a paradigmatic raft constituent, actively contributed to the biogenesis and maturation of autophagic vacuoles. In particular, fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses revealed that this ganglioside interacts with phosphatidylinositol 3-phosphate and can be detected in immature autophagosomes in association with LC3-II as well as in autolysosomes associated with LAMP1. Hence, it appears as a structural component of autophagic flux. Accordingly, we found that autophagy was significantly impaired by knocking down ST8SIA1/GD3 synthase (ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 1) or by altering sphingolipid metabolism with fumonisin B1. Interestingly, exogenous administration of GD3 ganglioside was capable of reactivating the autophagic process inhibited by fumonisin B1. Altogether, these results suggest that gangliosides, via their molecular interaction with autophagy-associated molecules, could be recruited to autophagosome and contribute to morphogenic remodeling, e.g., to changes of membrane curvature and fluidity, finally leading to mature autolysosome formation.