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Metastases are the leading cause of cancer-related deaths, and their origin is not fully elucidated. Recently, studies have shown that extracellular vesicles (EVs), particularly small extracellular vesicles (sEV), can disrupt the homeostasis of organs, promoting the development of a secondary tumor. However, the role of sEV in brain endothelium and their association with metastasis related to breast cancer is unknown. Thus, this study aimed to investigate sEV-triggered changes in the phosphorylation state of proteins on the surface of brain endothelial cells, as they form the first barrier in contact with circulating tumor cells and EVs, and once identified, to modulate its interactors and effects from this through different functional assays. We used the most aggressive breast cancer cell line, MDA-MB-231, and its brain-seeking variant, MDA-MB-231-br. From these cells, small and large extracellular vesicles were harvested to treat hCMEC/D3 cells, an immortalized cell line from the human brain microvasculature. Higher levels of phosphorylation of VEGFR1 and VEGFR2 were found in hCMEC/D3 cells treated with MDA-MB-231-br sEV. By computational analysis, the Valosin-Containing Protein (VCP) was predicted to be an important sEV cargo affecting the VEGFR2 intracellular trafficking, validated by western blotting analysis. Then, VCP was modulated by cell transfection or chemical inhibition in hCMEC/D3 cells and assessed in different functional in vitro assays evidencing a significant effect on the functionality of these cells. Thus, this study demonstrates that the VCP-containing sEVs induce modifications at different phosphor sites of VEGFR2 and effectively modulate the state of brain microvascular endothelial cells.