As a result of cytotoxic compounds against cancer cell lines from natural sources, senven compounds were isolated from the leaf and twig of Acer okamotoanum Nakai. The compounds (1-7) were identified as ethyl gallate (1), methyl gallate (2), gallic acid (3), trans resveratrol-3-O-β-D-glucopyranoside (4), acertannin (5), nikoenoside (6), and fraxin (7) by physicochemical and spectroscopic data (including mp, UV, IR, MS, 1H-NMR, 13C-NMR, DEPT, and HMBC) in comparison with those of published papers. All the compounds were tested for their cytotoxic activity against L1210, HL-60, K562, and B16F10 cancer cell lines in vitro by MTT assay method. Compounds 1-3 and 5 showed cytotoxic activity against all tested cell lines with IC50 values ranged from 12.5 to 72.2 μM. Of the compounds, methyl gallate (2) exhibited the most potent cytotoxic activity against L1210, HL-60, K562, and B16F10 tumor cells with IC50 values of 12.5, 48.3, 22.8, and 22.8 μM, respectively. Other compounds did not show any cytotoxic activity against four cancer cell lines.