Objective: This study aimed to optimiz the extraction process of polysaccharide from the seeds of Trichosanthes Kirilowii Maxim and investigate the primary structure and hypoglycemic activity of polysaccharide. Methods: The extraction process of polysaccharide from seed of Trichosanthes Kirilowii Maxim was optimized by hot water extraction. The homogenous polysaccharide (WHP-I) was purified by dextran gel G-150, and its structure was characterized. The hypoglycemic activity of Trichosanthes Kirilowii seed polysaccharide was investigated by in vitro and in vivo experiments. Results: The optimal conditions for extracting polysaccharide from Trichosanthes Kirilowii seed by hot water extraction were 80℃, 100 min, 1:25 solid-liquid ratio (g/mL), and two times of extraction. Verification test results showed that the extraction rate of polysaccharide was 17.54%, and the optimal scheme was scientific and reasonable. The primary structure analysis showed that WHP-I had an average molecular weight of 47.65 kDa and contained five sugar residues: →3)-Araf-(1→,→6)-Galp-(1→,→4)-Glcp-(1→, D-Glcp-(1→,and →4,6)-D-Glcp-(1→. The surface of WHP-I was flat, with pore structure and fine particles attached to the surface. The results of in vitro hypoglycemic experiment showed that the IC50 value of WHP-I inhibited α-glucosidase was 16.837 mg/mL. In vivo experiments showed that WHP-I has the potential to slow the organ damage caused by T2DM. After 4 weeks of administration, the blood glucose levels of M-WHP-I group and H-WHP-I group were reduced by 32.13% and 42.27%, respectively. Moreover, WHP-I could repair the kidney and liver of diabetic mice. Conclusion: The optimized process of polysaccharide from the seeds of Trichosanthes Trichosanthes Kirilowii was reasonable and feasible, with clear structure and obvious hypoglycemic activity.