Anoectochilus roxburghii, a traditional Chinese medicinal herb, has been widely used for treating numerous chronic diseases. In this study, a polysaccharide from A. roxburghii (ARPs-p) was purified by anion exchange and size exclusion chromatography. The structural characteristics of ARPs-p were systematically investigated for the first time via numerous chromatographic techniques, periodic acid oxidation, Smith degradation, methylation analysis, FTIR spectroscopy and 1D/2D NMR spectroscopy. The results showed that ARPs-p is a heteropolysaccharide with a molecular weight of 97 kDa; it consists of 97.75% glucose, 1.2% galactose and trace amounts of galacturonic acid, and its backbone structure is composed of →3)-β-D-Glcp-(1→ with some branching points at O-6 linked to non-reducing end units or 6-O-linked Glcp units. Furthermore, streptozotocin (STZ)-induced diabetic mouse experiments suggested that ARPs-p has excellent antihyperglycemic, antioxidant and antihyperlipidemic activities, in which 1,3-β-D-glucan, the main component of ARPs-p, plays a vital role.