Skeletal muscle is the principal site for postprandial glucose utilization and augmenting the rate of glucose utilization in this tissue may help to control hyperglycemia associated with diabetes mellitus. Here, we explored the effect of Deoxyandrographolide (DeoAn) isolated from the Andrographis paniculata Nees on glucose utilization in skeletal muscle and investigated its antihyperglycemic effect in vivo in streptozotocin-induced diabetic rats and genetically diabetic db/db mice. In L6 myotubes, DeoAn dose-dependently stimulated glucose uptake by enhancing the translocation of glucose transporter 4 (GLUT4) to cell surface, without affecting the total cellular GLUT4 and GLUT1 content. These effects of DeoAn were additive to insulin. Further analysis revealed that DeoAn activated PI-3-K- and AMPK-dependent signaling pathways, account for the augmented glucose transport in L6 myotubes. Furthermore, DeoAn lowered postprandial blood glucose levels in streptozotocin-induced diabetic rats and also suppressed the rises in the fasting blood glucose, serum insulin, triglycerides and LDL-Cholesterol levels of db/db mice. These findings suggest the therapeutic efficacy of the DeoAn for type 2 diabetes mellitus and can be potential phytochemical for its management.