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AMP-activated protein kinase (AMPK) plays an important role in mediating energy metabolism and is controlled mainly by two upstream kinases, LKB1 or Ca(2+)/calmodulin-dependent protein kinase kinase-β (CaMKKβ). Previously, we found that baicalin, one of the major flavonoids in a traditional Chinese herb medicine, Scutellaria baicalensis, protects against the development of hepatic steatosis in rats feeding with a high-fat diet by the activation of AMPK, but, the underlying mechanism for AMPK activation is unknown. Here we show that in two LKB1-deficient cells, HeLa and A549 cells, baicalin activates AMPK by α Thr-172 phosphorylation and subsequent phosphorylation of its downstream target, acetyl CoA carboxylase, at Ser-79, to a similar degree as does in HepG2 cells (that express LKB1). Pharmacologic inhibition of CaMKKβ by its selective inhibitor STO-609 markedly inhibits baicalin-induced AMPK activation in both HeLa and HepG2 cells, indicating that CaMKKβ is the responsible AMPK kinase. We also show that treatment of baicalin causes a larger increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), although the maximal level of [Ca(2+)](i) is lower in HepG2 cells compared to HeLa cells. Chelation of intracellular free Ca(2+) by EDTA and EGTA, or depletion of intracellular Ca(2+) stores by the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin abrogates baicalin-induced activation of AMPK in HeLa cells. Neither cellular ATP nor the production of reactive oxygen species is altered by baicalin. Finally, in HeLa cells, baicalin treatment no longer decreases intracellular lipid accumulation caused by oleic acid after inhibition of CaMKKβ by STO-609. These results demonstrate that a potential Ca(2+)/CaMKKβ dependent pathway is involved in the activation of AMPK by baicalin and suggest that CaMKKβ likely acts as an upstream kinase of AMPK in response to baicalin.