Here, we studied whether RAB27B affects the EV cargo by using click chemistry to label newly synthesized proteins in EVs, and MRPS‐based quantitative proteomics analysis, and we demonstrate that TGF‐β upregulates the quantity of protein cargo and alters the protein composition in EVs in breast cancer cells primarily mediated by the downregulation of RAB27B expression. Here, RAB27B is linked to breast cancer.