We previously showed that prostaglandin F(2α) (PGF(2α)) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in part via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of AMP-activated protein kinase (AMPK), an intracellular energy sensor, in PGF(2α)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2α) time-dependently induced the phosphorylation of the AMPK α-subunit. Compound C, an inhibitor of AMPK, dose-dependently suppressed PGF(2α)-stimulated IL-6 release. Compound C reduced the PGF(2α)-induced acetyl-CoA carboxylase phosphorylation. In addition, PGF(2α)-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The IL-6 mRNA expression induced by PGF(2α) was markedly reduced by compound C. Downregulation of the AMPK α1-subunit by short interfering RNA (siRNA) significantly suppressed the PGF(2α)-stimulated IL-6 release. PGF(2α)-induced phosphorylation of p38 MAP kinase was inhibited by compound C, which failed to affect the p44/p42 MAP kinase phosphorylation. These results strongly suggest that AMPK regulates PGF(2α)-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts.