Aggregation of the high affinity receptor for IgE (FcεRI) on mast cells by antigen and IgE complex induces release of chemical mediators, leading to acute allergic inflammation. We recently found that 3-O-(2,3-dimethylbutanoyl)-13-O-decanoylingenol (DBDI), purified from the Euphorbia kansui L., inhibits degranulation in rat basophilic leukemia 2H3 cells upon aggregation of the FcεRI. In the present study, we demonstrated that the DBDI significantly inhibits release of β-hexosaminidase, synthesis of eicosanoids, and mobilization of intracellular Ca²⁺ in the bone marrow-derived mouse mast cells stimulated with IgE and antigen. Furthermore, we revealed that phosphorylation of Syk, phospholipase C-γ₂, and extracellular signal-related kinase 1/2 is significantly suppressed in the DBDI-treated mast cells. These findings suggest that the DBDI may have a therapeutic potential for allergic diseases by inhibiting intracellular signaling pathways for activation and chemical mediator release in mast cells.