To establish a systematic method for the extraction, purification, characterization and antitumor activity study of polysaccharide from artificially cultivated Anoectochilus roxburghii (wall.) Lindl. (AC-ARPS). High-speed counter current chromatography with two-phase aqueous systems was successfully applied to purify AC-ARPS after one-step separation. The purity of the AC-ARPS obtained by phenol/sulfuric acid method was 95.01%. The chemical structures of AC-ARPS were identified by a series of analytical methods including high-performance liquid chromatography and liquid chromatography with mass spectrometry. High-performance liquid chromatography and liquid chromatography with mass spectrometry indicated that AC-ARPS was mainly composed of mannose, ribose, glucose, galactose and arabinose with a molar ratio of 1.00:8.47:47.30:1.17:1.19. AC-ARPS is a homogeneous polysaccharide with a molecular weight of 25 681 Da. The antitumor effect of AC-ARPS was evaluated on lung cancer A549, osteosarcoma 143B, rat adrenal pheochromocytoma PC 12, breast cancer MCF-7, acute leukemia HL 60, chronic leukemia K562, colon cancer SW620, esophageal cancer OE 19, liver cancer HepG2, and neuroglioma U251 cells in vitro. AC-ARPS showed the best inhibitory effect on OE 19 cells, and the IC value was 5.67 ± 0.831 μmol/L. Fluorescence analysis and flow cytometry results showed that AC-ARPS induced apoptosis and G2/M phase arrest in OE 19 cells.