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Pancreatic ductal adenocarcinoma (PDAC) is a fatalmalignant cancer with extremely poor prognosis and high mortality. Genome wide studies show that Slit/Robo signaling pathway takes a major effect in the oncogenesis and progression of pancreatic cancer. However, the function and mechanism of ROBO2 in the development of PDAC remains unclear. In present study, we use Western blot and real-time polymerase chain reaction (RT-PCR) to detect the expression of ROBO2 in pancreatic cell lines. Cell proliferation,Transwellmigration and invasion were conducted inAsPC-1, MIA PaCa-2 and PANC-1cell lines. RNA sequencing, bioinformatics analysisand Western blot were used to explore its mechanism and potential target molecules. The expression of ROBO2 in 95 tumor tissues was detected by immunohistochemistry. ROBO2 expression was downregulated in PDAC cell lines and tissue samples. A high expression of ROBO2 was associated with better prognosis. Upregulation of ROBO2 inhibited PDAC cell proliferation, migration, and invasion. However, we found theoppositeresults in the ROBO2 downregulation group. In addition, the function of ROBO2 on cell proliferation was further affirmed by the animal model. Finally, the results of RNA sequencing indicated that ROBO2 partly promoted the antitumor activity by inhibiting ECM1 in PDAC. Our work suggests that ROBO2 inhibits tumor progression in PDAC and may serve as a predictive biomarker and therapeutic target in PDAC. [ABSTRACT FROM AUTHOR]