Lipid synthesis and storage in adipose tissues contribute to energy homeostasis, which is inversely regulated by activated AMP-activated protein kinase (AMPK). Here, I report that Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta) negatively regulates lipogenesis in adipocytes through its phosphorylation of AMPK. Specifically, the glucagon-activated CaMKKbeta/AMPK cascade inhibits acetyl-CoA carboxylase (ACC) activity through the phosphorylation of ACC both in vitro and in vivo. Fasting increases, whereas refeeding decreases, the phosphorylation of AMPK and ACC in CaMKKbeta+/+, but not in CaMKKbeta-/- mice. Under ad libitum feeding, the increased expression of sterol regulatory element binding protein-1c (SREBP-1c)-mediated lipogenic gene is concurrent with decreased phosphorylation of AMPK-regulated ACC in white adipose tissue (WAT) of CaMKKbeta-/- mice. In line with the inhibitory role of CaMKKbeta in lipid synthesis and storage, CaMKKbeta-/- mice exhibit increased body weight, adiposity, and adipocyte hypertrophy, although their appetite is comparable with the CaMKKbeta+/+ littermates. Additionally, metformin, an AMPK activator, does not increase the phosphorylation of AMPK and ACC in WAT of CaMKKbeta-/- mice. Thus, the CaMKKbeta/AMPK signaling is an important molecular event in physiological and pharmacological (e.g., metformin) regulation of adiposity.