AMP-activated protein kinase (AMPK) is a key regulator of glucose and fatty acid homeostasis. In muscle cells, AMPK stimulates mitochondrial fatty acid oxidation and ATP production. The thyroid hormone T3 increases cellular oxygen consumption, and is considered to be a major regulator of mitochondrial activities. In this study, we examined the possible involvement of AMPK in the stimulatory action of T3 on mitochondria. Treatment of C2C12 myoblasts with T3 rapidly led to phosphorylation of AMPK. Acetyl-CoA carboxylase, a direct target of AMPK, was also phosphorylated following T3 treatment. Similar results were obtained with 3T3-L1, FRTL-5 and HeLa cells. Stable expression of T3 receptor (TR)- or TR in Neuro2a cells enhanced this effect of T3, indicating the involvement of TRs. Because HeLa cells express only Ca²⁺/calmodulin-dependent protein kinase kinase- (CaMKK), one of two known AMPK kinases, it was suggested that the effect of T3 is mediated by CaMKK. Indeed, experiments using a CaMKK inhibitor, STO-609, and an isoform-specific small interfering RNA demonstrated the CaMKK-dependent phosphorylation of AMPK. Furthermore, T3 was found to rapidly induce intracellular Ca²⁺ mobilization in HeLa cells, and a Ca²⁺-chelator, BAPTA, suppressed T3- as well as ionomycin-dependent phosphorylation of AMPK. In addition, T3-dependent oxidation of palmitic acids was attenuated by BAPTA, STO-609 and the small interfering RNA for CaMKK, indicating that T3-induced activation of AMPK leads to increased fatty acid oxidation. These results demonstrate that T3 non-transcriptionally activates AMPK via intracellular Ca²⁺ mobilization and CaMKK activation, thereby stimulating mitochondrial fatty acid oxidation.