The study identifies CaMKKs as AMPK kinases (AMPKKs) in cells lacking LKB1. AMPK is a key regulator of cellular metabolism, activated by phosphorylation of its α subunit at Thr-172. This phosphorylation is mediated by AMPKKs, with LKB1 being a major AMPKK. However, other AMPKKs, including CaMKKα and CaMKKβ, were also identified. In HeLa and A549 cells, treatments with mannitol, 2-deoxyglucose, and ionomycin activated AMPK through αThr-172 phosphorylation, which was inhibited by the CaMKK inhibitor STO-609. In vitro, STO-609 inhibited AMPKK activity in HeLa cell lysates, with an IC50 comparable to known CaMKK isoforms. RNA interference experiments confirmed that CaMKKα and CaMKKβ are functional AMPKKs, as their knockdown reduced AMPK activity and phosphorylation. In LKB1-deficient cells, AMPK activation by ionomycin and 2-deoxyglucose was not impaired, indicating that CaMKKs function as AMPKKs in these cells. These findings suggest that CaMKKs are important AMPKKs in regulating AMPK activity in vivo, beyond LKB1. The study highlights the roles of CaMKKs in AMPK regulation, particularly in response to metabolic stress and intracellular Ca²+ changes. The results indicate that CaMKKs may play significant roles in AMPK regulation in various tissues and cell types.The study identifies CaMKKs as AMPK kinases (AMPKKs) in cells lacking LKB1. AMPK is a key regulator of cellular metabolism, activated by phosphorylation of its α subunit at Thr-172. This phosphorylation is mediated by AMPKKs, with LKB1 being a major AMPKK. However, other AMPKKs, including CaMKKα and CaMKKβ, were also identified. In HeLa and A549 cells, treatments with mannitol, 2-deoxyglucose, and ionomycin activated AMPK through αThr-172 phosphorylation, which was inhibited by the CaMKK inhibitor STO-609. In vitro, STO-609 inhibited AMPKK activity in HeLa cell lysates, with an IC50 comparable to known CaMKK isoforms. RNA interference experiments confirmed that CaMKKα and CaMKKβ are functional AMPKKs, as their knockdown reduced AMPK activity and phosphorylation. In LKB1-deficient cells, AMPK activation by ionomycin and 2-deoxyglucose was not impaired, indicating that CaMKKs function as AMPKKs in these cells. These findings suggest that CaMKKs are important AMPKKs in regulating AMPK activity in vivo, beyond LKB1. The study highlights the roles of CaMKKs in AMPK regulation, particularly in response to metabolic stress and intracellular Ca²+ changes. The results indicate that CaMKKs may play significant roles in AMPK regulation in various tissues and cell types.