AMP-activated protein kinase (AMPK) is recognized as a main regulator of energy homeostasis. Osteocalcin (OC), which is produced specifically by mature osteoblasts, is stored in bone matrix, strongly binds to hydroxyapatite and is released into the circulation, has been recognized as a marker of bone metabolism. It has recently been shown that OC released from osteoblasts influences energy metabolism as a hormone. We previously reported that triiodothyronine (T3) stimulates the synthesis of OC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether AMPK participates in T3-stimulated OC synthesis in osteoblasts. T3 time-dependently induced the phosphorylation of the AMPKα-subunit (Thr-172), whereas T3 failed to induce the phosphorylation of AMPKα-subunit (Ser-485), AMPKβ-subunit (Ser-108) and AMPKβ-subunit (Ser-182). Both the release and the mRNA expression of OC induced by T3 were significantly inhibited by compound C, an AMPK inhibitor. Compound C suppressed the T3-induced phosphorylation of acetyl-CoA carboxylase, a direct substrate of AMPK. T3-stimulated OC release was significantly reduced in AMPK-knockdown cells using AMPK-siRNA. These results strongly suggest that AMPK positively regulates T3-stimulated OC synthesis in osteoblasts.