An extract from Artemisia dracunculus L. (termed PMI-5011) improves glucose homeostasis by enhancing insulin action and reducing ectopic lipid accumulation, while increasing fat oxidation in skeletal muscle tissue in obese insulin resistant male mice. A chalcone, DMC-2, in PMI-5011 is the major bioactive that enhances insulin signaling and activation of AKT. However, the mechanism by which PMI-5011 improves lipid metabolism is unknown. AMPK is the cellular energy and metabolic sensor and a key regulator of lipid metabolism in muscle. This study examined PMI-5011 activation of AMPK signaling using murine C2C12 muscle cell culture and skeletal muscle tissue. Findings show that PMI-5011 increases Thr172-phosphorylation of AMPK in muscle cells and skeletal muscle tissue, while hepatic AMPK activation by PMI-5011 was not observed. Increased AMPK activity by PMI-5011 affects downstream signaling of AMPK, resulting in inhibition of ACC and increased SIRT1 protein levels. Selective deletion of DMC-2 from PMI-5011 demonstrates that compounds other than DMC-2 in a “DMC-2 knock out extract” (KOE) are responsible for AMPK activation and its downstream effects. Compared to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, the phytochemical mixture characterizing the KOE appears to more efficiently activate AMPK in muscle cells. KOE-mediated AMPK activation was LKB-1 independent, suggesting KOE does not activate AMPK via LKB-1 stimulation. Through AMPK activation, compounds in PMI-5011 may regulate lipid metabolism in skeletal muscle. Thus, the AMPK-activating potential of the KOE adds therapeutic value to PMI-5011 and its constituents in treating insulin resistance or type 2 diabetes.