A methanol extract of the roots of Sophora flavescens Aiton, which is used as a crude drug Sophora Root (Sophorae Radix; "Kujin" in Japanese) and is listed in the Japanese Pharmacopoeia XIX, exerted antiallergic effects on ear passive cutaneous anaphylaxis (PCA) reactions in mice. From the extract, we isolated three previously undescribed prenylflavonoids, kujinfulavanonols A (1) and B (2) and kujinflavanone A (3), along with 34 known compounds (4-37). The absolute stereostructures of 1-3 were established by comparing experimental and predicted ECD data as well as through DP4+ analysis using the calculated 13C NMR chemical shifts. Among the isolates, principal prenylflavonoide sophoraflavanone G (4) and matrine-type alkaloid oxymatrine (30) showed in vivo antiallergic activity at a dose of 50 mg/kg, p.o. To determine the mechanisms of this antiallergic effect, hyaluronidase inhibitory activity and antigen-induced degranulation inhibitory activity were examined in vitro. The results showed that i) sophoraflavanone G (4, IC50 = 34.1 μM), isoxanthohumol (12, 49.1 μM), and kushenols X (14, 22.9 μM) and I (15, 40.1 μM) and ii) sophoraflavanone G (4, IC50 = 48.9 μM), leachianone A (5, 28.9 μM), kushenol U (6, 32.9 μM), (-)-kurarinone (7, 90.5 μM), (2S)-2'-methoxykurarinone (8, 34.4 μM), and isoxanthohumol (12, 58.5 μM), (2R)-3β,7,4'-trihydroxy-5-methoxy-8-prenylflavanone (17, 50.8 μM), and (2R)-3α,7,4'-trihydroxy-5-methoxy-8-prenylflavanone (19, 66.6 μM) were the active constituents.