Tumor-derived extracellular vesicles (EVs) have been associated with cancer progression and metastasis due to the transfer of bioactive molecules in between the tumor microenvironment. Although Transforming Growth Factor-β (TGF-β) signaling associated molecules have been implicated as cargo of EVs, the role of TGF-β pathway on EV’s biology to promote tumorigenesis is poorly understood. Hence, we aimed to describe the impact of TGF-β signaling on biogenesis and functional transfer of cargo from tumor-derived EVs induced by TGF-β (EV+TGF-β1). To this end, EV-fractions enriched in CD81 or Cholerae toxin B chain (CTB) were isolated from the vesicular secretome fraction (VSF) of human breast cancer and lung adenocarcinoma cells and further characterized.