Abstract Exosomes are extracellular vesicles containing nucleic acids, proteins, and lipids of approximately 100 nm, which are released from cancer cells to create a microenvironment favorable for their survival. Epidermal growth factor receptor pathway substrate 8 (Eps8), an exosomal protein, is reportedly associated with metastasis. We investigated the relationship between Eps8 in the serum exosomes of patients with pancreatic cancer and clinicopathological factors and examined the usefulness of exosomal Eps8 as a biomarker for pancreatic cancer. Methods: Serum exosomes of 93 patients with pancreatic disease (64 cases of pancreatic cancer, 12 cases of chronic pancreatitis, and 17 cases of IPMN) were extracted via ultracentrifugation, and Eps8 in exosomes were evaluated by western blotting. We analyzed 1) the clinicopathological factors of patients with pancreatic cancer, 2) the Eps8 positive rate in each disease, 3) the sensitivity, specificity, and positive predictive value of Eps8 for the diagnosis of pancreatic cancer, 4) the relationship between Eps8 and clinicopathological factors, and 5) compared and discussed between exosomal Eps8 and serum CA19-9. Results: The median patient age was 72 years (47–85), with 35 male and 29 female patients included in the study. The median body mass index was 20.8 kg/m2 (12–26.7 kg/m2). 1) Site Ph33/Pbt31, cStageIV/III/II/I: 28/11/14/11; 2) pancreatic cancer 59.4%, chronic pancreatitis 33%, IPMN 12%; 3) sensitivity 59.4. 4) In patients with pancreatic cancer, Eps8-positive patients had significantly higher ALT (p = 0.03), significantly less cancer (p = 0.04), lower IPMN complication rates, and more chronic pancreatitis and autoimmune diseases than the exosomal Eps8-negative patients. Stage I 64% (7/11), Stage II 36% (5/14), Stage III 64% (7/11) Stage IV 68% (19/28) 5) Exosomal Eps8 positivity (64%) was higher than serum CA19-9 positivity (45%) in Stage I. We also found some patients were negative for serum CA19-9 but positive for exosomal Eps8. Conclusion: Exosomal Eps8 has a higher positive rate in Stage I than serum CA19-9. Diagnosis with exosomal Eps8 may improve diagnostic performance for pancreatic cancer. Citation Format: Kazunao Hayashi, Atsunori Tsuchiya, Shuji Terai. Exosomal protein Eps8 as a new biomarker for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B006.