Extracellular vesicles (EVs) are membrane-delineated particles secreted by most types of cells under both normal and pathophysiological conditions. EVs are believed to mediate intercellular communication by serving as carriers of different bioactive ingredients, including proteins, nucleic acids and lipids. Glycoconjugates are complex molecules consisting of covalently linked carbohydrate with proteins or lipids. These glycoconjugates play essential roles in the sorting of vesicular protein and the uptake of small extracellular vesicles (30-100 nm, sEVs) into recipient cells. Glycosphingolipids (GSLs), one subtype of glycolipids, which are ubiquitous membrane components in almost all living organisms, are also commonly distributed on sEVs. However, the study of functional roles of GSLs on sEVs are far behind than other functional cargos. The purpose of this review is to highlight the importance of GSLs on sEVs. Initially, we described classification and structure of GSLs. Then, we briefly introduced the essential functions of GSLs, which are able to interact with functional membrane proteins, such as growth factor receptors, integrins and tetraspanins, to modulate cell growth, adhesion and cell motility. In addition, we discussed analytical methods for studying GSLs on sEVs. Finally, we focused on the function of GSLs on sEVs, including regulating the aggregation of extracellular α-synuclein (α-syn) or extracellular amyloid-β (Aβ) and influencing tumor cell malignancy.