Small extracellular vesicles (sEVs) are particles secreted from cells that play vital roles both in normal physiology and in human disease. sEVs are highly enriched in tetraspanin proteins, such as CD9 and CD63, and contain tetraspanin-enriched membrane microdomains involved in loading of sEVs with macromolecule cargoes and in sEV biogenesis. However, the precise roles of individual tetraspanins in sEV biogenesis and cargo loading remain poorly understood. Here, we report that CD9 negatively regulated CD63 trafficking to tetraspanin-enriched microdomains and its subsequent packaging into sEVs, whereas CD63 had no discernable effect on CD9 localization or packaging. Using super resolution microscopy of individual vesicles, we showed that CD9 governs the fraction of sEVs that are loaded with CD63. Interestingly, CD9-dependent suppression of CD63 packaging was rescued by pharmacological blockade of endocytosis. Together, our data support a model where CD9 contributes to the regulation and secretion of CD63 in an endocytosis-dependent manner to reprogram the contents of sEVs and tetraspanin-enriched microdomains.