-Lipoic acid (ALA) widely exists in foods and is an anti-diabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 µM of ALA increased the activity and phosphorylation of AMPK at Thr¹⁷². The phosphorylation of AMPK substrate, acetyl CoA carboxylase (ACC) at Ser⁷⁹ was also increased. No difference in ATP, AMP, and the calculated AMP/ATP ratio was observed ps. Since the upstream AMPK kinase, LKB1, requires an alteration of AMP/ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased intracellular calcium concentration (P<0.05), showing that ALA may activate AMPK through enhancing calcium/calmodulin-dependent protein kinase kinases (CaMKK) signaling. Indeed, chelation of intracellular free calcium by BAPTA-AM abolished ALA-induced activation of AMPK and in turn phosphorylation of ACC at Ser⁷⁹. Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser⁷⁹. In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, siRNA was used to silence CaMKK which abolished the ALA induced AMPK activation. This data shows that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.