Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy. Overexpression of CaMKK2 suppressed C2C12 myoblast proliferation and differentiation, while inhibition of CaMKK2 had opposite effect. We also found that CaMKK2 is able to activate AMPK in C2C12 myocytes. Inhibition of AMPK could attenuate the effect of CaMKK2 overexpression, while AMPK agonist could abrogate the effect of CaMKK2 knockdown on C2C12 cell differentiation and proliferation. These results suggest that CaMKK2 functions as an AMPK kinase in muscle cells and AMPK mediates the effect of CaMKK2 on myoblast proliferation and differentiation. Our data also indicate that CaMKK2 might inhibit myoblast proliferation through AMPK-mediated cell cycle arrest by inducing cdc2-Tyr15 phosphorylation and repress differentiation through affecting PGC1α transcription. Lastly, we show that overexpressing CaMKK2 in the muscle of mice via electroporation impaired the muscle regeneration during freeze-induced injury, indicating that CaMKK2 could serve as a potential target to treat patients with muscle injury or myopathies. Together, our study reveals a new role for CaMKK2 as a negative regulator of myoblast differentiation and proliferation and sheds new light on the molecular regulation of muscle regeneration.