<p>Glycosphingolipids (GSLs) are regulators of membrane organization and trafficking. According to the glycolipid-lectin (GL-Lect) driven endocytosis model, GSLs are part of a mechanism in which they are used by oligomeric lectins from pathogens or cells to build tubular endocytic pits from which clathrin-independent endocytic carriers detach by friction-driven scission. This review revisits the unique architectural logic of GSLs and explores how this underpins mechanisms for endocytic trafficking and signaling. We examine notably how GSLs orchestrate retrograde trafficking from the plasma membrane to the Golgi apparatus and subsequent polarized secretion in specialized contexts such as migrating cells, epithelial tissues, and immune synapses. Despite their importance, GSL functions have remained difficult to explore, because of the scarcity of minimally perturbing, chemically defined tools. We survey recent development in GSL probe design and discuss principles for next-generation tools that retain native function while enabling mechanistic dissection. Finally, we discuss how these advances are reshaping therapeutic strategies, with implications for vaccine delivery, cancer targeting, and lysosomal storage disorders. Collectively, this review reframes GSLs as substrates of membrane organization, with vast untapped potential in both cellular membrane biology and translational medicine.</p>