Background: Schisandrol A, a lignan with anticancer effects, is one of the representative components that identifies Schisandra chinensis. Objective: A method for purifying schisandrol A from the stems of S. chinensis was established using an octadecylsilyl (ODS) column combined with preparative high-performance liquid chromatography (HPLC). Materials and Methods: Crude extracts obtained from the stems of S. chinensis using 70% ethanol were separated on an AB-8 macroporous resin column and then eluted with a graded ethanol series. After 70% methanol was used in an ODS column separation, preparative HPLC was used for subsequent purification. The structure was identified by electrospray ionization-mass spectrometry, 1 H-nuclear magnetic resonance, and 13 C-nuclear magnetic resonance. HepG2 and Bel-7402 hepatocellular carcinoma cell lines were used for toxicological evaluation. Results: 21.4 mg of schisandrol A with a purity of 95.2% were collected. The cytotoxicity of the ODS-purified sample and schisandrol A were significantly stronger than that of a resin-purified sample. Conclusion: Schisandrol A was successfully extracted from the stems of S. chinensis and separated with an ODS column combined with preparative HPLC. The samples obtained during the purification process showed different levels of cytotoxicity on the HepG2 and Bel-7402 hepatocellular carcinoma cell lines.