Extracellular vesicles (EVs) comprise two main classes - exosomes and shed microvesicles (sMVs) with distinct mechanisms of biogenesis. Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100-600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). We show that sMVs, unlike exosomes, are ALIX-, TSG101-, CD63- and CD9-. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures. sMVs from primary tumour cell lines are enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while metastatic tumour-derived-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts and exhibit fibroblast invasion capability, suggesting a role of sMVs in intercellular communication. We report, for the first time a comprehensive analysis of a hitherto undescribed subpopulation of EVs. We anticipate our in vitro findings will be a starting point for more sophisticated studies aimed at elucidating the biochemical and functional properties of EV subpopulations in vivo. Furthermore, the emerging roles of specific EV subpopulations in the tumour microenvironment we believe will alter our view of cancer biology and undoubtedly present new targets for therapeutic intervention.