Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell‑type specific, and molecules embedded in exosomes can be useful fluid‑based clinical biomarkers. To identify proteins with metastatic marker potential, we conducted a comparative exosomal proteome analysis using human pancreatic cancer cell lines derived from metastasis, ascites, and primary tumors. Metastatic potential of cell lines was assessed by migratory and invasive activities. A pancreatic cancer cell line from metastasis (SU.86.86) revealed 23‑fold and 20‑fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa‑2 cell line derived from primary tumor cells. Liquid chromatography‑mass spectrometry‑based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis‑derived SU.86.86 cells. Comparison of 12 pancreatic cancer cell lines derived from different stages of malignancy revealed a strong relationship between exosomal Eps8 protein levels and cell motile activities (migration: r=0.85, P=4.2x10‑4; invasion: r=0.60, P=3.2x10‑2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0x10‑2; invasion: r=0.51, P=9.2x10‑2). It was therefore concluded that exosomal Eps8 protein levels were correlated with the migratory cell potential of human pancreatic cancer cells, indicating that exosomal Eps8 has the potential to be a metastatic biomarker for human pancreatic cancer.