AMP-activated protein kinase (AMPK) is activated by upstream kinases and negatively regulated by protein phosphatases. Intracellular calcium mediates protein phosphatase 2A (PP2A), which is in a heterotrimeric complex with the PR72 subunit. The PR72 subunit contains two calcium-binding sites formed by EF hands. Our previous study has shown that chronic calcium exposure decreases AMPK activity. To define the specific molecular mechanism whereby calcium can deactivate AMPK, activities of AMPK and PP2A were analyzed in C2C12 muscle cell cultures and skeletal muscle tissues from mutant pigs possessing the AMPKγ3-mutation or the ryanodine receptor (RyR1) calcium gating mutation, or both. C2C12 myotubes treated with calcium releasing agent (caffeine) for 10h decreased (P<0.05) AICAR-induced AMPK activity to control levels and this negative effect was eliminated by ryanodine receptor stabilizer, dantrolene. Interestingly, muscle from pigs with the RyR1 mutation and C2C12 cells administered with 10h caffeine showed higher (P<0.05) PP2A activity compared to controls. More importantly, the inhibitory effect of caffeine on AMPK activity was attenuated by the PP2A inhibitor, calyculin A or siRNA induced knockdown of PP2A. These data show the inhibitory effect of chronic calcium on AMPK activity is exerted through the activation of PP2A.