Updated project metadata. The calcium-calmodulin (Ca2+-CaM) dependent protein kinase kinase-2 (CaMKK2) is activated by increases in intracellular Ca2+ and is a key regulator of cellular and wholebody energy metabolism. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma and metabolic derangements induced by a high-fat diet, therefore elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cyclic AMP (cAMP)-dependent protein kinase (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs Ca2+-CaM activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495. We also report the crystal structure of 14-3-3z bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and non-canonical (Ser100) 14-3-3 consensus sites on CaMKK2 co-operate to bind 14-3-3 proteins. Our findings provide a detailed mechanistic insight into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.