The pancreas morphology of transgenic mice that overexpress transforming growth factor-β1 (TGF-β1) in the pancreas resembles partially morphological features of chronic pancreatitis, such as progressive accumulation of extracellular matrix (ECM). Using this transgenic mouse model, we characterized the composition of pancreatic fibrosis and involved fibrogenic mediators. On day 14 after birth, fibrotic tissue was mainly composed of collagen type I and III. At this time, mRNA levels of TGF-β1 were increased. On day 70, the ECM composition was expanded by increased deposition of fibronectin, whereas connective tissue growth factor, fibroblast growth factor (FGF)-1, and FGF-2 mRNA expression levels were elevated in addition to TGF-β1. In parallel, the number of pancreatic stellate cells (PSC) increased over time. In vitro, TGF-β1 stimulated collagen type I expression but not fibronectin expression in PSC, in contrast to FGF-2, which stimulated both. This confirms that TGF-β1 mediates pancreatic fibrosis through activation of PSC and deposition of collagen type I and III at early time points. Furthermore, this points to an indirect mechanism in which TGF-β regulates pancreatic ECM assembly by induction of additional growth factors.