The study investigates the biogenesis of exosomes, focusing on the highly enriched exosomal tetraspanins CD81, CD9, and CD63. The findings suggest that endocytosis inhibits the vesicular secretion of these proteins and triggers their destruction, particularly for CD9 and CD81. The research also reveals that syntenin, an exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking its endocytosis. Additionally, high-level expression of CD63, which binds to the clathrin adaptor AP-2 subunit mu2, inhibits endocytosis and induces the plasma membrane accumulation and vesicular secretion of CD63. The results indicate that the vesicular secretion of exosome marker proteins primarily occurs through an endocytosis-independent pathway, challenging the prevailing paradigm of exosome biogenesis.The study investigates the biogenesis of exosomes, focusing on the highly enriched exosomal tetraspanins CD81, CD9, and CD63. The findings suggest that endocytosis inhibits the vesicular secretion of these proteins and triggers their destruction, particularly for CD9 and CD81. The research also reveals that syntenin, an exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking its endocytosis. Additionally, high-level expression of CD63, which binds to the clathrin adaptor AP-2 subunit mu2, inhibits endocytosis and induces the plasma membrane accumulation and vesicular secretion of CD63. The results indicate that the vesicular secretion of exosome marker proteins primarily occurs through an endocytosis-independent pathway, challenging the prevailing paradigm of exosome biogenesis.