Hulless barley grass may confer many health benefits attributed to its bioactive functional components, such as polysaccharides. Here, a hot water soluble polysaccharide was extracted from hulless barley grass, and its chemical characterization and in vitro anti-cancer activities were investigated. The yield of hulless barley grass polysaccharide (HBGP) was 2.3%, and the purity reached 99.1% with a polydispersity index (PDI) of 1.11 after purification by a diethylaminoethyl cellulose (DE-32) column and an S-400 high resolution (HR) column. The molecular weight and number-average molecular weight of HBGP were 3.3 × 104 and 2.9 × 104 Da, respectively. The monosaccharide composition of HBGP included 35.1% galactose, 25.6% arabinose, 5.5% glucose, and 5.3% xylose. Based on infrared spectrum analysis, HBGP possessed pyranose and galactose residues. In addition, this water-soluble polysaccharide showed significant cell proliferation inhibitory effects against cancer cell lines HT29, Caco-2, 4T1, and CT26.WT in a dose-dependent manner, especially for HT29 (the half-inhibitory concentration IC50 value = 2.72 mg/mL). The results provide a basis for the development and utilization of hulless barley grass in functional foods to aid in preventing cancer. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.