Changes within interstitial stromal compartments often accompany carcinogenesis, and this is true of prostate cancer. Typically, the tissue becomes populated by myofibroblasts that can promote progression. Not all myofibroblasts exhibit the same negative influence, however, and identifying the aggressive form of myofibroblast may provide useful information at diagnosis. A means of molecularly defining such myofibroblasts is unknown. We compared protein profiles of normal and diseased stroma isolated from prostate cancer patients to identify discriminating hallmarks of disease-associated stroma. We included the stimulation of normal stromal cells with known myofibroblast inducers namely soluble TGFβ and exosome-associated-TGFβ and compared the function and protein profiles arising. In all 6-patients examined, diseased stroma exhibited a pro-angiogenic influence on endothelial cells, generating large multicellular vessel-like structures. Identical structures were apparent following stimulation of normal stroma with exosomes (5/6 patients), but TGFβ-stimulation generated a non-angiogenic stroma. Proteomics highlighted disease-related cytoskeleton alterations such as elevated Transgelin (TAGLN). Many of these were also changed following TGFβ or exosome stimulation and did not well discriminate the nature of the stimulus. Soluble TGFβ, however triggered differential expression of proteins related to mitochondrial function including voltage dependent ion channels VDAC1 and 2, and this was not found in the other stromal types studied. Surprisingly, Aldehyde Dehydrogenase (ALDH1A1), a stem-cell associated protein was detected in normal stromal cells and found to decrease in disease. In summary, we have discovered a set of proteins that contribute to defining disease-associated myofibroblasts, and emphasise the similarity between exosome-generated myofibroblasts and those naturally arising in situ.