The purpose of this study was to investigate the pharmacological effect of fraction of Atractylodes lancea (Thunb.) DC. (A. lancea) extract. In this study, we isolated different polarity fractions, including petroleum ether (PE), ethyl acetate, n-butanol, and the remaining H2O fractions from the water extract of A. lancea. The antigastric cancer properties of the different fractions in BGC-823 and SGC-7901 cells were evaluated. Apoptotic cells were treated with PE fraction and stained with Hoechst 33342 and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide. The cell cycle was analyzed via flow cytometry. The main compounds of PE fraction were determined by HPLC-ESI-MS. Results of this study showed that the PE fraction of A. lancea inhibited the growth of BGC-823 and SGC-7901 cells in a dose- and time-dependent manner. The morphological and mitochondrial transmembrane potential changes suggested that the cells showed preliminary apoptosis characteristics after treatment with the three different polarities. The main compounds of PE fraction include two sesquiterpene compounds: eudesm-4(15),7-diene-9α,11-diol and eudesm-4(15)-ene-7a,11-diol; three sesquiterpene lactone compounds: atractylenolid I, atractylenolid III and 3-β-acetyl-atractylenolid III and one polyacetylenic compound: 4,6,12-tetradecatriene-8,10-diyne-1,3,14-triol.