Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, being predicted to become the second leading cause of cancer-related death by 2030. Chronic pancreatitis is a risk factor for PDAC and both diseases are characterized by a strong desmoplastic response, comprised of activated myofibroblasts and immune cell infiltrates. Genomic aberrations in the SLIT-ROBO pathway are frequent in PDAC. Nevertheless, their role in the pancreas is unclear. We have used an integrative approach combining the study of murine models and PDAC patients with the objective of unraveling the function of the SLIT-ROBO signaling pathway in pancreatic disease. RNA expression of SLIT-ROBO genes was analyzed in murine normal pancreas, pancreatitis and PDAC. Primary cell cultures and experimental pancreatitis were studied using pancreas-specific Robo2 (Pdx1-Cre;Robo2F/F) and whole-body Slit1 (Slit1-/-) knockout mice. Gene and protein expression were assessed in a cohort of PDAC patients (n=109). In mouse pancreatitis and PDAC, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Pdx1Cre;Robo2F/F pancreatic cell cultures showed increased activation of Robo1-positive myofibroblasts and induction of TGF-β and Wnt pathways. Likewise, induction of pancreatitis in Pdx1Cre;Robo2F/F mice enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. Similar results were obtained using Slit1-/- animals. Moreover, TGF-β inhibition using galunisertib treatment suppressed Robo2-mediated effects in the microenvironment. In patients, ROBO2 expression is overall low in PDAC, while ROBO1 is variably expressed in epithelium and high in the stroma. ROBO1 expression is correlated with markers of activated stroma, Wnt and TGF-β pathways. ROBO2low;ROBO1high subpopulation of patients present the poorest survival rates. In conclusion, Robo2 acts nonautonomously as a stroma suppressor gene by restraining myofibroblast activation and inflammation in the pancreatic microenvironment. ROBO1/2 expression is prognostic in PDAC patients and may guide therapy with TGF-β inhibitors or immunotherapies, currently being tested in clinical trials for advanced pancreatic cancer.