The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help develop therapeutic interventions. Serotonin (5-hydroxytryptamine, 5-HT) has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, namely inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar de-differentiation and cell cycle progression during the regenerative phase were investigated over the course of two weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect clonal regeneration of mature acinar cells. Serotonin was however required for acinar de-differentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes, delayed formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in the progenitor-based regeneration, as it promoted acinar cell de-differentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas activated by zymogen secretion. Our findings indicate that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting de-differentiation of acinar cells.