Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1‐phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P₂). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P₂ antagonism on portal hypertension was examined. Intravenous infusion of the S1P₂ antagonist, JTE‐013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P₂ antagonist did not alter portal vein pressure and mean arterial pressure in control sham‐operated rats. Rho kinase activity in the livers was enhanced in bile duct‐ligated rats compared to sham‐operated rats, and this enhanced Rho kinase activity in bile duct‐ligated livers was reduced after infusion of the S1P₂ antagonist. S1P₂ messenger RNA (mRNA) expression, but not S1P₁ or S1P₃, was increased in bile duct‐ligated livers of rats and mice and also in culture‐activated rat hepatic stellate cells. S1P₂ expression, determined in S1P mice, was highly increased in hepatic stellate cells of bile duct‐ligated livers. Furthermore, the increase of Rho kinase activity in bile duct‐ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P mice. Conclusion: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P₂. The S1P₂ antagonist merits consideration as a novel therapeutic agent for portal hypertension. (HEPATOLOGY 2012)