1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) is a gallotannin isolated from various plants such as Galla Rhois. In a previous study, it was reported that PGG has anti-allergic effects by inhibiting interleukin (IL)-4 signaling in B cells. However, the effect of PGG on basophilic cells remains unclear. Therefore, the aim of this study was to investigate the inhibitory effect of PGG on mitogen and calcium ionophore-induced allergic responses. PGG had no effect on proliferation and cytotoxicity of RBL-2H3 cells. PGG significantly suppressed cell degranulation (histamine and β-hexosaminidase) as well as inflammatory cytokine production such as IL-4 and tumor necrosis factor-α. The underlying mechanism of PGG on these anti-allergic actions was correlated with inhibition on translocation of nuclear factor-κB from the cytosol to nucleus. These data suggest that PGG is a potentially effective functional compound for prevention of allergic diseases.