Azurocidin (AZU1) is an antimicrobial protein secreted by neutrophils that acts as a chemoattractant for monocytes and macrophages and a permeabilizer of vascular endothelial cells. We previously identified AZU1 to be specifically present in extracellular vesicles (EVs) obtained from renal cell carcinoma (RCC) tissues. Here, we examined the relationship between N-linked glycosylation and AZU1 loading into small EVs (SEVs). Inhibition of N-linked glycosylation by introducing mutations in three glycosylation sites inhibited AZU1 loading into SEVs. Furthermore, SEVs released from AZU1-wild-type cells increased the Ca concentration in endothelial cells and the endothelial permeability, whereas SEVs released from AZU1-mutant cells had no significant effect. Anti-AZU1 antibodies diminished the effect of SEVs on endothelial cell sheets. Collectively, we found that N-linked glycosylation of AZU1 directs its loading into SEVs, thereby enabling AZU1-positive SEVs to function as potent permeabilizers of endothelial cells and leading to enhanced transendothelial migration of RCC cells.