1. In response to pancreatic injury and in cell culture, pancreatic stellate cells (PSCs) are transformed ('activated') into highly proliferative myofibroblast-like cells, which express alpha-smooth muscle actin (alpha-SMA), and produce type I collagen and other extracellular matrix components. There is accumulating evidence that activated PSCs play important roles in pancreatic fibrosis and inflammation. 2. The small GTP-binding protein Rho has emerged as an important regulator of the actin cytoskeleton and cell morphology through the downstream effector Rho kinase (ROCK). But, the roles of Rho-ROCK pathway in PSCs are unknown. Here, we examined the effects of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and HA-1077 (fasudil), specific inhibitors of ROCK, on the activation of PSCs. 3. PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P. The actin cytoskeleton was analyzed by phalloidin staining. Expression of RhoA and ROCK was examined by immunostaining and Western blotting. Effects of Y-27632 and HA-1077 on alpha-SMA expression, platelet-derived growth factor-induced proliferation and chemotaxis, and collagen production were assessed. 4. Culture-activated PSCs developed a well-spread cell shape, with extended stress fiber formation. PSCs expressed RhoA, ROCK-1, and ROCK-2. 5. Y-27632 caused disassembly of stress fibers. Y-27632 and HA-1077 inhibited alpha-SMA expression, proliferation, chemotaxis, and type I collagen production in culture-activated PSCs. 6. In addition, Y-27632 and HA-1077 inhibited spontaneous activation of freshly isolated PSCs in culture on plastic. 7. These findings suggest a role of Rho-ROCK pathway in the activation process of PSCs by regulating the actin cytoskeleton, and a potential application of Rho-ROCK pathway inhibitors for the treatment of pancreatic inflammation and fibrosis.