This study explored the structural characteristics and in vitro hypoglycemic activity of polysaccharides from the stems of Dendrobium nobile Lindl.. Two homogeneous polysaccharides, DNP-6 and DNP-7, were obtained by water extraction followed by alcohol precipitation and subsequent successive anion exchange and gel column chromatography. Their structures were analyzed by high performance liquid chromatography (HPLC), ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR). The results showed that DNP-6 and DNP-7 were neutral polysaccharides with molecular mass of 2.70 × 10³ and 1.23 × 10³ Da, containing 90.45% and 93.49% total sugar and 2.28% and 1.29% protein, respectively. They were mainly composed of glucose and mannose, with molar ratios of 6.76:1 and 6.63:1, respectively. The glycosidic bonds were mainly of α-configuration, with a small amount of β-pyranose. One-dimensional and two-dimensional NMR results showed that the main chain composition of DNP-6 and DNP-7 was: →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-β-D-Manp-(1→4)-α-D-Glcp/β-D-Glcp. In addition, the results of in vitro glucose consumption and insulin resistance tests showed that both polysaccharides had good hypoglycemic activity, and DNP-6 possessed stronger activity than DNP-7.