Pancreatic stellate cells (PSCs) play a central role in the development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of normal pancreas. We here evaluated the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Mitogen-activated protein (MAP) kinase protein levels and a- smooth muscle actin (-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-1 (TGF-1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative polymerase chain reaction (PCR) and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and -SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAP kinase. Treatment of PSCs with a MAP kinase kinase (MEK) inhibitor and p38 MAP kinase inhibitor suppressed pressure-induced cell proliferation and -SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-1 secretion, collagen type I mRNA expression and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs, and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.