The leaves of Mitragyna speciosa Korth (Rubiaceae) have been used in folk medicine for its unique medicinal properties. This study examined the water, methanolic and crude alkaloidal extracts from M. speciosa leaves and its major constituent mitragynine for the enhancement of glucose transport. Cellular uptake of radioactive 2-deoxyglucose was determined in rat L8 myotubes. Involving signalling pathway was determined with the specific inhibitors. Cell cytotoxicity was monitored by lactate dehydrogenase assay. Protein levels of glucose transporters (GLUTs) were measured by Western blotting. The results show that test samples significantly increased the rate of glucose uptake. The uptake was associated with increase in GLUT1 protein content. Co-incubation with insulin had no additional effect, but the cellular uptake was decreased by wortmannin and SB 203580, specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK), respectively. It is concluded that the increased glucose transport activity of M. speciosa is associated with increases in activities of the key enzymes dependent to the insulin-stimulated glucose transport for its acute action, and increases in the GLUT1 content for its long-term effect. This study demonstrated the effect of M. speciosa in stimulating glucose transport in muscle cells, implicating the folkloric use of M. speciosa leaves for treating diabetes.