The key components of Grifola frondosa polysaccharide F2 that contribute to lowering blood sugar were examined. A Sephacryl S-200 column was used for the isolation and purification of G. frondosa polysaccharide F2 to obtain polysaccharides F21 and F22. These polysaccharides were then subjected to structural analyses and assessment of their hypoglycemic activity. Structural analyses revealed that polysaccharide F21 is composed of fucose, galactose, glucose, and mannose, and its molecular weight is approximately 17.9 ku. F22 consists of mannose and glucose, with a molecular weight of approximately 3.16 ku. Animal experiments revealed that F21 (i.g.) administration significantly reduced fasting blood glucose and triglyceride levels in T2DM mice by 16.36% and 26.53% (P<0.05), respectively. F22 (i.g.) administration significantly decreased the low density lipoprotein level in T2DM mice by 31.48% (P<0.01). F21 (i.p.) administration decreased the fasting blood glucose level in T2DM mice by 22.32% (P<0.05). Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) decreased by 32.96% (P<0.001), 36.12% (P<0.05), and 42.35% (P<0.001), respectively, compared with the diabetes group. Hepatic malondialdehyde (MDA) content decreased by 42.95% (P<0.001), whereas superoxide dismutase (SOD) and catalase activities are increased by 36.08% (P<0.05) and 24.34% (P<0.01), respectively. F22 (i.p.) administration decreased liver ALP by 32.38% (P<0.01), increased SOD activity by 37.32% (P<0.05), and reduced MDA content by 68.16% (P<0.001). These results demonstrate that the active component of G. frondosa polysaccharide F2 is F21, which has the ability to alleviate symptoms in T2DM model mice. This study provides a theoretical framework for the development and application of G. frondosa polysaccharides in food and medicine, highlighting their diverse prophylactic benefits.