Isoflavones are known to possess immunomodulating and antiallergic activities. Previously we identified novel isoflavone methyl-glycosides (daidzein 7-O-β-d-glucoside 4″-O-methylate (CDGM), glycitein 7-O-β-D-glucoside 4″-O-methylate (CGLM), genistein 7-O-β-D-glucoside 4″-O-methylate (CGNMI) and genistein 4'-O-β-D-glucoside 4″-O-methylate (CGNMII)) from Cordyceps militaris grown on germinated soybeans (GSC). The biological activity of novel isoflavone methyl-glycosides, however, remains unknown. In this study, CGNMII showed the strongest inhibition of degranulation. Additionally, the release of interleukin (IL)-4 and tumor necrosis factor (TNF)-α was decreased by CGNMII in antigen-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism of CGNMII, we examined whether it affected levels of signaling molecules responsible for degranulation. The levels of activated Lyn, Syk, PLCγ1 and LAT proteins were reduced in CGNMII treated RBL-2H3 cells. CGNMII also inhibited the activation of AKT and ERK1/2 proteins. These results suggest that CGNMII might be used as a therapeutic agent for allergic diseases.