CaMKK1 and CaMKK2 kinases regulate key physiological and pathological processes such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation and cellular energy homeostasis, and promote cell survival. We have elucidated the structural basis of the inhibition of both CaMKK kinases by the regulatory proteins 14-3-3. Our results show that binding of the 14-3-3 protein to both CaMKK1 and CaMKK2 prevents their interaction with calmodulin, a signaling molecule that is essential for their activity. Comparison of the structures of the 14-3-3 complexes with CaMKK also revealed that the catalytic center of CaMKK1 is blocked by the C-terminal helices of the 14-3-3 protein. Our findings may help in the development of new drugs targeted to inhibit CaMKK kinases.