The dry flower buds of Sophora japonica L. are used as a hemostatic agent in traditional Chinese medicine. In the comet assay, aqueous extracts of S. japonica decreased and increased the tail length significantly in cultured human lymphoblastoid WTK1 cells exposed to UV in the absence and presence of DNA repair inhibitors, respectively. The extract did not affect the tail length in methyl methanesulfonate-exposed cells. In the present study, the aqueous extract of the flower buds of S. japonica was separated by repeated column chromatography, yielding four types of flavonoid glycosides. Among them, only rutin, similar to the extract, decreased and increased the tail length significantly in WTK1 cells exposed to UV in the absence and presence of DNA repair inhibitors hydroxyurea (10 mM) and cytosine-1-β-D-arabinofuranoside (1.8 mM), respectively.  The genotoxicity-suppressing effect of rutin was further studied using the micronucleus test. Rutin significantly decreased the frequency of micronucleated binucleate cells in UV-exposed WTK1 cells but did not affect this frequency in UV-exposed XPL3KA (Xeroderma pigmentosum group C) cells. These results suggest that the anti-genotoxic potential of rutin is due to an enhanced incision step of global genome repair (GGR) sub-pathways in nucleotide excision repair (NER). Herein, we show that S. japonica exhibits heretofore unknown anti-genotoxic potential against UV by enhancing the incision of GGR sub-pathways in NER, and that its anti-genotoxic component is rutin.