question: Extracellular Vesicles (EVs) are a heterogenous population of particles that play an important role in cell-cell communication in physiological and pathophysiological situations. In this study we reveal that the peptidyl prolyl isomerase Cyclophilin A (CypA) is enriched in cancer-derived EVs from a range of haematopoietic malignancies. CypA-enriched blood cancer EVs were taken up by normal monocytes independent of EV surface trypsin-sensitive proteins and potently stimulated pro-inflammatory MMP9 and IL-6 secretion. Further characterisation revealed that CypA is intravesicular, however, it is not present in all EVs derived from the haematopoietic cells, instead, it is predominantly located in high density EVs with a range of 1.15–1.18 g/ml. Furthermore, loss of CypA expression in haematological cancer cells attenuates high density EV-induced pro-inflammatory MMP9 and IL-6 secretion from monocytes. Mechanistically, we reveal that homozygous loss or siRNA knockdown of CypA expression significantly reduced the secretion of EVs in the range of 100–200 nm from blood cancer cells under normal and hypoxic conditions. Overall, this work reveals a novel role for CypA in cancer cell EV biogenesis.