AbstractCell‐derived extracellular vesicles (EVs) are evolutionary‐conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs), and shed midbody remnants (lEVs/sMB‐Rs). sEVs/Exos are of endosomal pathway origin, microparticles (lEVs/MPs) from plasma membrane blebbing and shed midbody remnants (lEVs/sMB‐Rs) arise from symmetric cytokinetic abscission. Here, we isolate sEVs/Exos, lEVs/MPs, and lEVs/sMB‐Rs secreted from human isogenic primary (SW480) and metastatic (SW620) colorectal cancer (CRC) cell lines in milligram quantities for label‐free MS/MS‐based proteomic profiling. Purified EVs revealed selective composition packaging of exosomal protein markers in SW480/SW620‐sEVs/Exos, metabolic enzymes in SW480/SW620‐lEVs/MPs, while centralspindlin complex proteins, nucleoproteins, splicing factors, RNA granule proteins, translation‐initiation factors, and mitochondrial proteins selectively traffic to SW480/SW620‐ lEVs/sMB‐Rs. Collectively, we identify 39 human cancer‐associated genes in EVs; 17 associated with SW480‐EVs, 22 with SW620‐EVs. We highlight oncogenic receptors/transporters selectively enriched in sEVs/Exos (EGFR/FAS in SW480‐sEVs/Exos and MET, TGFBR2, ABCB1 in SW620‐sEVs/Exos). Interestingly, MDK, STAT1, and TGM2 are selectively enriched in SW480‐lEVs/sMB‐Rs, and ADAM15 to SW620‐lEVs/sMB‐Rs. Our study reveals sEVs/Exos, lEVs/MPs, and lEVs/sMB‐Rs have distinct protein signatures that open potential diagnostic avenues of distinct types of EVs for clinical utility.