Normal cerebral vasculature is characterized by high selectivity, low permeability and low transcytosis rates, ensuring proper brain physiology. Our group has demonstrated that extracellular vesicles isolated from brainseeking MDA-MB-231 cells (Br-EVs) breach the intact BBB in vivo and significantly increase the incidence of breast-to-brain metastasis, suggesting a dysfunction of the cerebral microvasculature. To elucidate the mechanisms by which Br-EVs modify the BBB and promote breast-to-brain metastasis, we utilized a quantitative global proteomic approach to assess changes in protein expression within murine cerebral microvessels post-Br-EV treatment. We employed label-free mass spectrometry-based quantitative proteomics to analyze the protein content of cerebral microvessels from the brains of mice treated with PBS, P-EVs or Br-EVs.