Extracellular vesicles (EVs), a generic term for any vesicles or particles that are released from cells, play an important role in modulating numerous biological and pathological events, including development, differentiation, aging, thrombus formation, immune responses, neurodegenerative diseases, and tumor progression. During the biogenesis of EVs, they encapsulate biologically active macromolecules (i.e., nucleotides and proteins) and transmit signals for delivering them to neighboring or cells that are located some distance away. In contrast, there are receptor molecules on the surface of EVs that function to mediate EV-to-cell and EV-to-matrix interactions. A growing body of evidence indicates that the EV surface is heavily modified with glycans, the function of which is to regulate the biogenesis and extracellular behaviors of EVs. In this chapter, we introduce the current status of our knowledge concerning EV glycosylation and discuss how it influences EV biology, highlighting the potential roles of EV glycans in clinical applications.