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Pancreatic stellate cells (PSCs) are important players in pancreatic fibrosis and are major contributors to the extracellular matrix proteins observed with the stromal response characteristic of pancreatic ductal adenocarcinoma (PDAC). Pancreatic stellate cells are also believed to secrete soluble factors that promote tumor progression; however, no comprehensive analysis of the PSC proteome in either the quiescent or the activated state has been reported.Using 2-dimensional tandem mass spectrometry and the RLT-PSC cell line, we present the first comprehensive study describing and comparing the quiescent and activated human PSC-secreted proteomes.Very few proteins are secreted in the quiescent state. In stark contrast, activated PSCs secreted a vast array of proteins. Many of these proteins differed from those secreted by PDAC-derived cell lines. Proteins associated with wound healing, proliferation, apoptosis, fibrosis, and invasion were characterized. Selected proteins were verified in human tissue samples from PDAC, dysplastic pancreas, and normal pancreas using Western blot analysis and immunohistochemical staining.Our study represents the first comprehensive analysis of proteins secreted by PSCs. These findings lay the foundation for characterizing PSC-derived proteins involved in stroma-tumor interactions and the promotion of pancreatitis and PDAC.