Objectives: Interleukin-36 (IL-36) is a recently described proinflammatory cytokine, characterized by the induction of inflammatory mediators. In the present study, we investigated the biological activity and the signal transduction of IL-36 alpha in human pancreatic myofibroblasts. Methods: The mRNA and protein expression of inflammatory mediators was evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of IL-36 alpha and its receptor in the pancreatic tissue was evaluated using immunohistochemical technique. Intracellular signaling pathways were evaluated using immunoblotting and specific small interference RNA-transfected cells. Results: Interleukin-36 alpha and its receptor complex IL-36R/IL-1RAcP were detected in fibrotic tissue of chronic pancreatitis. Interleukin-36 alpha dose-and time-dependently induced the mRNA expression and protein secretion of CXCL1, CXCL8, MMP-1, and MMP-3 from human pancreatic myofibroblasts. Interleukin-36 alpha assembled MyD88 adaptor proteins (MyD88, TRAF6, IRAK1, and TAK1) into a complex. Furthermore, IL-36 alpha induced the phosphorylation of mitogen-activated protein kinases and the activation of nuclear factor kappa B and activator protein 1. Mitogen-activated protein kinase inhibitors and small interference RNAs specific for nuclear factor kappa B and activator protein 1 significantly suppressed the protein secretion of inflammatory mediators induced by IL-36 alpha stimulation. Conclusions: It was suggested that IL-36 alpha plays an important role in the pathophysiology of inflammation and fibrosis in the pancreas via an autocrine function.