CD63 is a tetraspanin that functions as a master regulator of endosomal trafficking and extracellular vesicle biogenesis. Structurally, CD63 contains four transmembrane domains and localizes to both the plasma membrane and late endosomes/lysosomes 1. Primary to its function, CD63 acts as a cell surface receptor for TIMP1 and regulates integrin signaling through ITGB1 activation, promoting cell survival, adhesion, migration, and actin cytoskeleton reorganization via AKT and FAK/PTK2 pathways. CD63 also modulates VEGFA signaling through KDR/VEGFR2 internalization regulation. Mechanistically, CD63 operates as a lipid-sorting molecule that directs cholesterol into intraluminal vesicles of multivesicular endosomes, generating a mobilizable pool exported via exosomes 2. The protein undergoes clathrin-dependent endocytosis and enriches on intraluminal vesicles for exosomal secretion 1, with endocytosis inhibition promoting its vesicular secretion 3. CD63 expression is iron-regulated via the IRE-IRP system, influencing ferritin secretion in extracellular vesicles 4. ESCRT machinery components including HRS and TSG101 facilitate CD63-containing exosome secretion 5. Clinically, CD63-positive epididymosomes from human semen improve sperm function 6, suggesting therapeutic potential. However, CD63 plays only marginal roles in general protein sorting into extracellular vesicles 7, highlighting specialized rather than global EV composition functions.