Small membrane-derived extracellular vesicles have been proposed to participate in several cancer diseases, including breast cancer. We performed phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined 3 cell lines as models for breast cancer: MCF10A (non-metastatic), MCF7 (estrogen and progesterone receptor positive, metastatic) and MDA-MB-231 (triple-negative, highly metastatic). To obtain a comprehensive overview of the sEV phosphoproteme derived from each cell line, an effective enrichment phosphopeptide techniques with IMAC and TiO2, followed by LC-MS/MS was achieved. In total, 2003 phosphopeptides on which 207, 854 and 1335 have been identified in MCF10A, MCF7 and MDA-MB-231cell lines, respectively. These phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. These proteins are associated with larger number of diseases and the most common diseases are related to cancer. Among the phosphoproteins identified, we validated four enzymes associated to cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), Sirtuin-1 (SIRT1) and Sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential in early BC diagnostic and therapeutic applications.