In this study, polysaccharides from Atractylodes macrocephala Koidz (APA) which were soluble in alcohol were prepared, purified, analyzed the structure and investigated the antitumor activity in vitro cell experiment. Results of high-performance gel permeation chromatography (HPGPC), fourier-transform infrared spectroscopy (FT-IR), and gas chromatography (GC) showed that APA was a 2.1KDa neutral hetero polysaccharide composed of arabinose and glucose (molar ratio, 1.00:4.57) with pyranose rings and α-type and β-type glycosidic linkages. Results by MTT experiments showed that the proliferation inhibition was 74.63% in Eca109 cells treated with 2 mg/mL dose of APA. Annexin V/PI assay, Hoechst 33,258 staining, cell cycle distribution, rhodamine 123 dye assay and western blot assay clarified that APA could accelerate the apoptosis of Eca109 cells by mitochondrial pathway and stocked cells at S phase. These data indicated that APA is a promising potential candidate for therapeutic treatment of esophageal cancer.