In our program to synthesize a series of novel derivatives as potential analogs of honokiol for anti-tumor treatment, we have found that at least three of the derivatives of honokiol showed more potency to inhibit the proliferation of K562 leukemia cells and SPC-A1 adenocarcinoma cells. As a critical step to our further series synthesis of derivatives of honokiol, three derivatives of honokiol composed of two isomers and one compound with two formyl groups, which were hardly separated by common purification methods, needed to be rapidly separated and purified. The present work describes analytical and preparative high-speed counter-current chromatography (HSCCC) for the isolation and purification of these three C-formylation derivatives of honokiol, named 3'-formylhonokiol, 5-formylhonokiol and 3',5-diformylhonokiol, respectively. The solvent system for HSCCC separation was composed of hexane-ethyl acetate-methanol-water with the ratio of 1:0.4:1:0.4 (v/v). The one-step purification produced 157.8 mg, 121.6 mg and 21.2 mg of 3'-formylhonokiol, 5-formylhonokiol, 3',5-diformylhonokiol from crude sample of 400mg with purities of 98.6%, 99.2% and 99.6%, respectively, in an elution time of 2.5 h. The purities and structural identification were determined by HPLC, (1)H NMR, (13)C NMR and mass spectroscopy. Their anti-proliferation effects on K562, A549 and SPC-A1 cell lines were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.