Extracellular vesicles (EVs) are vital for information exchange between donor and recipient cells. When cells are stressed (e.g., by oxygen glucose deprivation, OGD), the complex information carried by the EVs is altered by the donor cells. Here, we aimed to analyze the proteomic differences between EVs derived from OGD-damaged cells and EVs derived from undamaged cells to explore the potential mechanisms by which EVs aggravate ischemic stroke (IS).EVs released by rat adrenal gland PC12 cells subjected to 0, 3, 6, or 12 h of OGD were isolated. The proteins from the EVs secreted by each of the OGD groups were profiled using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). We predicted the functions, pathways, and interactions of the differentially expressed proteins using Gene Ontology (GO), KEGG pathways, and STRING. We used parallel reaction monitoring (PRM) to validate our results.We identified several differentially expressed proteins in the OGD groups as compared to the controls: 170 proteins in the 3 h OGD EVs, 44 proteins in the 6 h OGD EVs, and 77 proteins in the 12 h OGD EVs (fold-change ≥1.5; p ≤ 0.05). These proteins were associated with oxidative stress, carbohydrate metabolism, protein synthesis and degradation, and thrombosis.We identified changes in protein expression in the EVs secreted by OGD-damaged cells, highlighting potential mechanisms by which EVs aggravate IS. Our results also suggested potential protein targets, which may be useful for the prevention and treatment of IS.