To the best of our knowledge, the current study is the first aimed at evaluating the physicochemical composition and proteomic profile of EVs from benign breast tumor, primary cells from malignant BC, and immortalized breast cell lines: a high-throughput proteomic approach was used to explore the EV proteome of two FADs, two distinct BC subtypes (i.e., HER2+ and TNBC), and the IBCC lines BT-549, MCF–10A, and MDA-MB-231. This evidence concerns the gene ERBB2 and breast cancer.