Extracellular vesicles (EVs) are membranous structures of variable size, shed from diverse cell types including highly invasive, fast migrating, amoeboid tumor cells. Silencing of the Diaphanous-related formin-3 (DIAPH3) protein in DU145 prostate cancer cells induces an amoeboid phenotype characterized by membrane blebbing and shedding of an atypically large population of EVs termed large oncosomes. We employed this cell system to characterize, for the first time, the global protein composition of large oncosomes and small EVs. The salient finding of this study, revealed by SILAC-based LC-MS/MS analysis was the identification of 20 unique proteins in large oncosomes and 7 in small EVs. Additionally, both types of EV were significantly enriched in proteins with cancer-related functions, suggesting that tumor interactions with the microenvironment are influenced by EV cargo. Proteins belonging to a series of functional classes, including GTPases, ESCRTs, RNA-binding proteins and membrane proteins, often identified in and which are sometimes considered specific for exosomes, appeared in both EV populations. Proteins such as CK18 and FN1, significantly enriched in large oncosomes, were selected to develop an assay to detect large oncosomes in the circulation. Finally, proteins over-represented in large oncosomes versus small EVs with at least a four-fold enrichment were significantly associated with cell migration, docetaxel resistance, angiogenesis, and prostate cancer bone metastasis, suggesting a distinct functional role in tumor progression for this larger class of EV. These observations indicate that, despite their origination from the same cell donors, the two distinct EV populations are enriched in different proteins and contribute diversified functions. These findings underscore the need for additional in-depth analyses to understand the unique contribution of subpopulations of EVs to tumor progression.