The secretory epithelial cells of the prostate gland use sophisticated vehicles in the form of prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. The aim of the present investigation was to conduct a proteomic analysis of metastasis-derived prostasomes. We investigated prostasomes from vertebral metastases of prostate cancer by 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) protein characterization. Twenty-five unique protein spots were identified by MALDI-TOF and another five proteins were determined by mass spectrometry (MS)/MS. Annexins A1, A3 and A5, as well as dimethylarginine dimethylaminohydrolase 1 were among the identified proteins. The annexins and dimethylarginine dimethylaminohydrolase 1 found in cancer-derived prostasomes can act, among others, as angiogenic factors and can increase the vascular development in the neighbourhood of the tumour. Cancer-derived prostasomes may play an important role in the interaction between tumour cells and their environment.