Ganoderma lucidum has long been used as a potent medicinal plant. In addition, recent studies showed that G. lucidum is used to prevent or treat various human diseases such as allergy, hepatopathy, hypertension, cancer and diabetes. In this study showed that ethanol extract from G. lucidum have potential inhibitory effects of glycerol 3-phosphat acyltransferase (GPAT) activity and increase of glucose uptake on skeletal muscle cells. Inhibition of GPAT, which catalyze the first step in de novo TAG synthesis, has been proposed as one of the drug targets for insulin resistance and type-2 diabetes. G. lucidum extract inhibited GPAT activity. Bioassay-guided fractionation and isolation of bio-active substances from ethanol extracts of G. lucidum were carried out by using chromatographic techniques and in vitro GPAT enzyme assay. One of bioactive molecules, ergosterol peroxide, was isolated and identified by physical and spectral properties. In this study showed that ethanol extract from G. lucidum and ergosterol peroxide have potential anti- diabetes effects through the increase of glucose uptake. This was as sociated with increased activity AMP-activated protein kinase(AMPK). AMPK is another regulatory protein in the glucose uptake pathway and energy metabolism. The result was that the increase of glucose uptake by G. lucidum extract might be mediated by AMPK.