Grapeseed extract (GSE) is a rich source of natural phenolic compounds and possesses various pharmacological activities, including antioxidant, anticarcinogenic and anti-inflammatory properties. However, effects of GSE on immunoglobulin (Ig) E-mediated allergic responses still remain elusive. In the present study, the effects of GSE on activation and degranulation of RBL-2H3 mast cells were investigated. GSE pretreatments (20–100 μg/ml) reduced IgE-antigen mediated release of β-hexosaminidase and histamine in RBL-2H3 cells. Additionally, GSE reversibly inhibited expression of FcεRI on RBL-2H3 cells. GSE treatments caused a significant elevation of intracellular cAMP levels, whereas the Ca2+ influx upon antigen stimulation was inhibited. Suppression on FcεRI expression together with decreased calcium uptake and increased cAMP level might be involved in attenuated degranulation of mast cells by GSE treatment. Our results suggest a possible pharmaceutical application of GSE in treating type I allergic diseases.