Exosomes are 40-100 nm diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly-purified material. Here, we describe an immunoaffinity-capture method using the colon epithelial cell specific A33 antibody to purify colorectal cancer cell (LIM1215) -derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled-coil, RAS and MIRO domains. A comparative protein profiling analysis of LIM1215, murine mast cell and human urinary derived-exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host-cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen (CEA), epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3 and 7), centrosomal protein 55kDa and ephrins-B1 and -B2. Further, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodelling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.