Our laboratory has previously demonstrated that Grifola frondosa polysaccharides (GFPs) showed hypoglycemic effects. This study aimed to investigate which polysaccharide-enriched fractions of GFPs were the main active constituents, and to disclose their hypoglycemic mechanism. F2 and F3 were obtained from GFPs and their hypoglycemic effects were investigated. Fasting serum glucose (FSG) levels, fasting serum insulin (FSI) levels and a homeostasis model assessment of insulin resistance (HOMA-IR) were measured, and the hepatic mRNA levels of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), protein tyrosine phosphatase-1B (PTP1B), phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B (PKB) were determined by a quantitative polymerase chain reaction (qPCR). The activity of IR and IRS-1 were determined by an enzyme-linked immunosorbent assay (ELISA), and their phospho-protein levels were analyzed with western blotting. F2 and F3 significantly decreased the levels of FSG, FSI and HOMA-IR compared with a diabetic control group (P < 0.05). F2 and F3 increased the activity and mRNA levels of IR, and the latter also increased the mRNA levels of IRS-1. As for the protein levels of phospho-IR and IRS-1, both F2 and F3 increased the protein levels of IR (Try 1361), but decreased IRS-1 (Ser307). In the PI3K/Akt pathway, F3 increased the mRNA levels of PI3K and Akt, however, F2 inhibited PTP1B expression. F2 and F3 are presumed to cause an improvement in insulin resistance, triggered by the reactivation of IR and IRS-1.