To isolate and characterize anti-tumor compounds through nuclear magnetic resonance (NMR) spectroscopy of ethyl acetate extract of R. japonicus Houtt root and their cytotoxic effects were evaluated in terms of measuring inhibition of growth of three cancer cell lines: human hepatoma cells (HepG2), cerebral glioma cells (U251) and ovarian carcinoma cells (SK-OV-3/DDP). The roots of R. japonicus Houtt were extracted with ethanol, and partitioned with petroleum ether, dichloromethane and ethyl acetate, respectively. The ethyl acetate extract was separated chromatographically using macroporous resin, positive and negative phase silica gel and high performance liquid chromatography. The structures of isolated compounds were elucidated by nuclear magnetic resonance (NMR) spectral data. Ten compounds were isolated form the ethyl acetate extract of R. japonicus roots. Their structures were determined as emodin (1), chrysophanol (2), aloe emodin (3), physcion (4), quercetin (5), resveratrol (6), β-sitosterol ( 7), 3β-acetoxy-28-hydroxyurs-12-ene (8), sucrose (9) and dibutyl phthalate (10). Among of them, compounds 1, 3, 4, 6 and 8 exhibited highly significant (p<0.05) cytotoxicities against HepG2 to a certain extent. Compounds 3-6 exhibited moderate cytotoxicities against U251 with IC 50 values of 19.8, 23.5, 23.8 and 17.8 µM, respectively. Compounds 1, 2 and 7 also exhibited cytotoxicities against U251 to a certain extent with IC 50 values of 48.3, 50.1 and 85.3 µM, respectively. Compounds 1-6 exhibited moderate cytotoxicities against (SK-OV-3/DDP) with IC 50 values of 62.8, 64.2, 31.3, 61.0, 47.4 and 42.0 µM, respectively.