Background & aims: Intestinal subepithelial myofibroblasts (SEMFs) is a specific population of cells involved in intestinal inflammation and carcinogenesis via an elaborate network of cytokines, chemokines and other inflammatory factors, including prostaglandin E₂ (PGE₂). Sphingosine-1-phosphate (S1P) has been implicated as an important mediator of inflammation and cancer, and in certain cell types increases COX-2 expression. In the present study, we aimed to assess involvement of S1P in COX-2 expression by SEMFs. Methods: Primary SEMFs were obtained from C57BL/6J mouse and their identity verified by fluorescent staining of specific marker proteins. Expression of S1P receptors 1, 2, 3 and sphingosine kinases 1 and 2 in SEMFs were determined by RT-PCR analysis. COX-2 expression and PGE₂ production were assayed by Western blotting and ELISA, respectively. COX-2 mRNA stability was assayed by Northern blotting. Results: S1P produced dose-dependent increase in COX-2 expression, resulting in increased PGE2 release from SEMFs. Using specific inhibitors, we show actions of p38, ERK, IKK and PKC were involved in S1P-induced COX-2 expression. On the other hand, p38 and PKC had lesser roles in IL-1-induced COX-2 expression. Inhibition of sphingosine kinase to block S1P production did not affect IL-1-induced COX-2 expression, but S1P amplified IL-1-induced p38 activation and COX-2 expression. PKC inhibition blocked S1P amplified COX-2 expression. S1P addition increased COX-2 mRNA stability. Conclusion: In SEMFs, S1P amplifies IL-1-induced COX-2 expression through increased mRNA stability. These observations point to involvement of S1P in activation of SEMFs that may contribute to intestinal inflammation and carcinogenesis.