We previously reported that sphingosine 1-phosphate induces heat shock protein 27 (HSP 27) via activation of phosphatidylinositol 3-kinase (PI3K)/Akt and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in sphingosine 1-phosphate-stimulated induction of HSP27 in MC3T3-E1 cells. Sphingosine 1-phosphate time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced sphingosine 1-phosphate-stimulated HSP27 induction, as well as MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, also suppressed sphingosine 1-phosphate-stimulated HSP27 induction. Y27632, as well as fasudil, attenuated sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase. However, Akt phosphorylation induced by sphingosine 1-phosphate was not affected by either Rho-kinase inhibitor. These results strongly suggest that Rho-kinase regulates sphingosine 1-phosphate-stimulated induction of HSP27 at a point upstream of p38 MAP kinase in osteoblasts.