We isolated three active constituents from an aqueous extract of spinach leaves by evaluating the inhibitory activity of IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells, and determined their chemical structures. These compounds, referred to as SO-1, SO-2 and SO-3, were assessed to be flavonol glucuronides with the same fundamental structure. Among them, SO-1 exhibited the most potent granulation-inhibitory activity at the concentrations of 0.5~10 µg/ml. This inhibitory effect was completely exhibited by its aglycon, which was prepared by the enzymatic treatment of β-glucuronidase and the chromatographic isolation. These compounds hardly showed cytotoxicity at the same concentration range in RBL-2H3 cell culture. To reveal its inhibitory mechanism, we examined the effects of SO-1 on the signal transduction of antigen-stimulated degranulation in the cells. Both SO-1 and its aglycon reduced the elevation of intracellular Ca 2+ concentration in a dose-dependent manner and also effectively inhibited the activation of tyrosine kinases such as Syk and Lyn, which underlay in the upstream of intracellular signal transduction followed by antigen stimulation. Furthermore, both SO-1 and its aglycon remarkably suppressed the passive cutaneous anaphylaxis (PCA) reaction induced in ICR mouse ear, as compared with the authentic anti-allergic drugs. In this PCA reaction, the aglycon exerted more potent inhibitory activity than SO-1, indicating that SO-1 also functions in vivo through the hydrolysis to the aglycon in the gastrointestinal tract. Together with these results, we conclude that spinach contains the promising anti-allergic flavonoid glucuronide, suggesting that this compound and its original vegetable may be beneficial to prevent or ameliorate allergic constitutions of humans.