Background: Mushrooms, including Astraeus spp., are known for hypoglycemic properties. Astraeus odoratus is a delicious edible mushroom, while Astraeus asiaticus is less popular due to its unpleasant texture. Both mushrooms have not been reported for the glucose uptake activities yet. Objectives: The aim of this study is to describe the enhancing of glucose uptake and related mechanisms in vitro of the extracts from two Astraeus spp. Materials and Methods: The extracts of A. odoratus and A. asiaticus (AO and AA, respectively) were assayed for the stimulation of glucose uptake in L6 myotubes. The mechanism of actions was proved by using specific inhibitors and determined for the expression of glucose transporters type 1 and 4 (GLUT1 and GLUT4) by quantitative real-time polymerase chain reaction and Western blotting. Results: The extracts of both mushrooms enhanced glucose uptake in the muscle cells L6 myotubes at the level of the function of GLUT1 and GLUT4, which involved the partial stimulation of their intrinsic activities through p38 mitogen-activated protein kinase and increased GLUT1 and GLUT4 protein levels. AO increased both GLUT1 and GLUT4 protein, while AA increased mainly GLUT4 protein and stimulated GLUT4 translocation through phosphatidylinositol 3-kinase. Conclusion: The results supported the hypoglycemic activity of A. odoratus and A. asiaticus and suggested their potential use for hypoglycemic purposes.