Insulin resistance is the main cause of type 2 diabetes, fatty liver and obesity. Our previous study found that mushroom polysaccharides improved insulin resistance in vitro, but the underlying mechanisms were still unknown. Thus, we investigate the hypoglycemic effects of polysaccharides from Gomphidiaceae rutilus fruiting bodies and their mechanisms. The total polysaccharides (AGRP) from Gomphidiaceae rutilus fruiting bodies and the neutral polysaccharide (AGRP-N) fraction both enhance insulin-stimulated glucose uptake in an autophagy-dependent manner in high glucose and fatty acid-treated hepatic cells, but not the acidic polysaccharide (AGRP-A) fraction. Further, we elucidate the oral hypoglycemic effects of polysaccharides on ob/ob mice. AGRP and AGRP-N lower blood glucose and improve insulin sensitivity. They inhibit liver lipid deposition, not only by activating AMPK to increase autophagy but also by increasing the expressions of PPARα and CPT-1a to enhance lipolysis. Our results provide a basis for the development of polysaccharides from Gomphidiaceae rutilus as a hypoglycemic healthy food.