Five FLAVONOID glycosides were isolated from then -butanol soluble fraction of the ethanolic extract of Rapistrum rugosum and their structures were assigned from 1H- and 13C-NMR spectra (DEPT) with 2D NMR as quercetin-3- O-a-L-rhamnopyranoside (1), quercetin-3- O-b-D-xyloside (2), quercetin, 3-O-a- L-arabinopyranoside, 7- O-a-L-rhamnopyranoside (3), kaempferol 3- O-a-L-arabinopyranoside, 7- O-a-L-rhamnopyranoside (4) and rutin (5). The SRB cytotoxic assay was used to investigate the antitumor activities of n -butanol extract, compound 3 and its hexaacetate 3a, for the first time. Compounds 3 and 3a showed cytotoxic activity against the human cancer cell line, namely, HepG2 (hepatocellular carcinoma cell line) with IC50 (concentration of compound required to reduce cell survival by 50%) 0.86 mg/mL and 3.50 mg/mL, respectively. These results proved that compound 3, the major FLAVONOID of the n -butanol soluble fraction, has significant cytotoxic activity compared with the standard antitumor drug doxorubicin (0.60 mg/mL).