Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis‐ and endocytosis‐like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ‐521) and metastatic (AZ‐P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC‐MS/MS and Western blot analyses, polyadenylate‐binding protein 1 (PABP1) was found to be predominantly abundant in AZ‐P7a exosomes. The amount of exosomal PABP1 in AZ‐P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin‐proteasome pathway (MG‐132 and PYR‐41). Treatment of AZ‐P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1‐immunoreactive products cleaved via a proteolysis‐like process. Taken together, these results suggest that AZ‐P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome‐mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.