The roots of Glycyrrhiza uralensis Fisch. were extracted with 30% aqueous ethanol (EtOH), and the concentrated extract was partitioned with n-hexane, chloroform ($CHCl_3$), ethyl acetate (EtOAc), n-butanol (n-BuOH), and $H_2O$, successively. From the $CHCl_3$ fraction, four flavonoids were isolated through the repeated silica gel ($SiO_2$), octadecyl silica gel (ODS), and Sephadex LH-20 column chromatographies (c.c.). According to the results of spectroscopic data including nuclear magnetic resonance spectrometry (NMR), electron ionization mass spectrometry (EI/MS), and infrared spectroscopy (IR), the chemical structures of the compounds were determined as glabrol (1), abyssinone II (2), glabridin (3), and isoliquiritigenin (4). The flavonoids were evaluated for cytotoxic effect against human cancer cell lines, HCT-116, HepG2, HeLa, SK-OV-3, SK-BR-3, MCF-7, and SK-MEL-5. Especially, glabrol (1) and glabridin (2) showed $IC_{50}$ values of lower than $25{mu}M$.