Abstract: After oil extraction, pumpkin seeds generate a large amount of waste meal, and polysaccharides are one of the main active components in pumpkin seed meal. To mitigate resource waste and maximize its value, pumpkin seed meal was used as raw material in this study to extract crude polysaccharides in large quantities by continuous phase transition extraction device. The crude polysaccharides were then subjected to precipitation using 95% ethanol and deproteinization using the Sevage method combined with enzymatic treatment, followed by further separation and purification via DEAE-52 and Experdex-75 columns. Finally, in vitro enzyme activity inhibition assays and 3T3-L1 adipocyte experiments were conducted to investigate and compare the hypoglycemic activity and underlying mechanism of the resulted various components. The study revealed that five polysaccharide fractions (PSP, PSP-1, PSP-1-1, PSP-1-2, PSP-1-3) were obtained after purification of pumpkin seed polysaccharides. The monosaccharide composition and molar ratio of psp-1-1, psp-1-2 and psp-1-3 were different. In vitro α-amylase and α-glucosidase inhibition assays showed PSP-1-1 exhibited the most potent effects with IC₅₀ values of 0.88 mg/mL and 3.92 mg/mL, respectively. Cellular experiments demonstrated PSP-1-1 significantly enhanced the cells’ ability to uptake glucose and markedly increased the high-density lipoprotein cholesterol (HDL-C) level to 257%, while reduced the levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) by 48.65%, 55.91% and 50%, respectively, suggesting it could alleviate cellular insulin resistance by improving glucose and lipid metabolism. It was hypothesized that the hypoglycemic mechanism of PSP-1-1 involved promoting the expression of genes in the insulin receptor substrate (IRS-1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) pathway, restoring normal cellular metabolism, and ultimately achieving hypoglycemic effect. This study enhances the comprehensive utilization value of pumpkin seed byproducts and provides theoretical support for the development of hypoglycemic functional foods with low toxicity and side effects.