Extracellular vesicles (EVs) are important mediators of intercellular communications in tumor microenvironment (TME). EV-mediated TGF-β signaling in the TME facilitates tumor metastasis and immune evasion by transferring TGF-β ligands and regulators to recipient cells. The mechanisms of how TGF-β affects cellular EV release and composition, however, are incompletely understood. Here, we systematically investigated the effects of TGF-β on EV release and protein composition. Here, we used quantitative proteomics to analyze how TGF-β regulates the protein composition of EVs by downregulating RAB27B expression.