Abstract Accumulating scientific evidences strongly support the importance of cancer-derived extracellular vesicles (EV) in organization of tumor microenvironment and metastatic niches, which are also considered as ideal tools for cancer liquid biopsy. To uncover the full scope of proteomic information packaged within EVs secreted directly from human colorectal cancer, we cultured surgically resected viable tissues and obtained tissue-exudative EVs (Te-EV). Our quantitative profiling of 6,307 Te-EV proteins and 8,565 tissue proteins from primary colorectal cancer and adjacent normal mucosa (n = 17) allowed identification of a specific cargo in colorectal cancer–derived Te-EVs, high-affinity cationic amino acid transporter 1 (CAT1, P = 5.0 × 10–3, fold change = 6.2), in addition to discovery of a new class of EV markers, VPS family proteins. The EV sandwich ELISA confirmed escalation of the EV-CAT1 level in plasma from patients with colorectal cancer compared with healthy donors (n = 119, P = 3.8 × 10–7). Further metabolomic analysis revealed that CAT1-overexpressed EVs drastically enhanced vascular endothelial cell growth and tubule formation via upregulation of arginine transport and downstream NO metabolic pathway. These findings demonstrate the potency of CAT1 as an EV-based biomarker for colorectal cancer and its functional significance on tumor angiogenesis. Implications: This study provides a proteome-wide compositional dataset for viable colorectal cancer tissue–derived EVs and especially emphasizes importance of EV-CAT1 as a key regulator of angiogenesis.