Updated publication reference for PubMed record(s): 33053347. The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries, generating considerably heterogeneous vesicle populations, i.e., exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here we discovered that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppressed the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. In the present study, to gain deeper insight into the molecular signatures of non-exosomal vesicles, we performed label-free quantitative proteomics of sEVs released from cells that were treated with or without 2-DG or NGI-1.