The rhizome of Polygonatum kingianum has been used as a traditional medicine in China. In this study, a novel polysaccharides (PKPs-1) was isolated from P. kingianum and characterized by its molecular weight, primary structure. The hypoglycemic activity of PKPs-1was investigated by in vitro assay with the HepG2 cell line and in vivo test using STZ-induced diabetic mice. Results showed that the average molecular weight of PKPs-1 was 14.05 kDa and is composed mainly of glucose and mannose. Methylation analysis indicated that this polysaccharides fraction consisted mainly of β1,2-link glucose. Besides, PKPs-1 exhibited significant anti-hyperglycemic activity on STZ-induced mice, improved insulin tolerance, and affected the metabolism of serum lipids. Results of real-time quantitative PCR (RT-PCR) showed that PKPs-1 significantly increased the expression of insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT), indicating that PKPs-1 regulates glucose metabolism by activating the PI3K/AKT signaling pathway. This study provides new insights for investigating the hypoglycemic effects of PKPs-1 and suggests that PKPs-1 could be a promising functional food or medicine for treating T2DM.