Sea buckthorn (Hippophae rhamnoides L.) is a deciduous shrub of the Elaeagnaceae family and is widely distributed in northern Eurasia. Sea buckthorn berry (SBB) has attracted attention for its use in many health foods, although its physiological function remains unknown. In this study, we investigated the inhibitory effect of SBB extract and its fractions on Type-I allergy using mast cell lines. Among these fractions, SBB fraction with the highest amount of antioxidant polyphenols significantly inhibited the release of chemical mediators such as histamine and leukotriene B4 (LTB4) from the stimulated mast cells. This fraction also inhibited the influx of calcium ions (Ca2+) and the phosphorylation of tyrosine residues in proteins, including spleen tyrosine kinase, which is associated with signal transduction during the release of chemical mediators. The active SBB fraction contained isorhamnetin as its major flavonol aglycon. Isorhamnetin inhibited histamine and LTB4 release from the stimulated cells and suppressed intracellular Ca2+ influx. These results indicate that isorhamnetin is the primary substance responsible for the antiallergic activity in SBB. In conclusion, SBB may alleviate Type-I allergy by inhibiting the release of chemical mediators from mast cells, and polyphenols may contribute to this effect.