Pancreatic ductal adenocarcinoma (PDAC), the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical to develop alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacologic approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up serotonin in a transport-mediated manner. Serotonin uptake promoted the activation of the small GTPase Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1), which is required for trans-differentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the serotonin transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of acinar-to-ductal metaplasia with the potential to stall initiation and progression of pancreatic carcinogenesis. This article is protected by copyright. All rights reserved.