We quantified 127 and 118 glycoproteins with different expressed N-glycosylation in the spermatozoa and seminal plasma of asthenozoospermic patients, with that more significant in the seminal plasma. GO analysis revealed these glycoproteins were highly enriched in extracellular vesicles (EVs) and many of them were pronounced EVs markers such as CD59, CD63 and Tetraspanins-8. Besides, we observed remarkable changes in N-glycosylations of Galectin-3 which is a glycoprotein important for fertilization located on sperm mainly transferred by EVs in seminal plasma. Furthermore, expression of some N-glycans in specific glycosites were strongly correlated to progressive motility.