The AMP-activated protein kinase (AMPK) is a critical regulator of energy balance at both the cellular and whole-body levels. Two upstream kinases have been reported to activate AMPK in cell-free assays, i.e., the tumor suppressor LKB1 and calmodulin-dependent protein kinase kinase. However, evidence that this is physiologically relevant currently only exists for LKB1. We now report that there is a significant basal activity and phosphorylation of AMPK in LKB1-deficient cells that can be stimulated by Ca2+ ionophores, and studies using the CaMKK inhibitor STO-609 and isoform-specific siRNAs show that CaMKKß is required for this effect. CaMKKß also activates AMPK much more rapidly than CaMKKa in cell-free assays. K+-induced depolarization in rat cerebrocortical slices, which increases intracellular Ca2+ without disturbing cellular adenine nucleotide levels, activates AMPK, and this is blocked by STO-609. Our results suggest a potential Ca2+-dependent neuroprotective pathway involving phosphorylation and activation of AMPK by CaMKKß.