AMP-activated protein kinase (AMPK) is a regulator of energy balance at both the cellular and the whole-body levels. Direct activation of AMPK has been highlighted as a potential novel, and possibly safer, alternative to treat type II diabetes and obesity. In this study, we aimed to design and characterize novel peptides that mimic the αG region of the α2 AMPK catalytic domain to modulate its activity by inhibiting interactions between AMPK domains or other interacting proteins. The derived peptides were tested in vivo and in tissue culture. The computationally predicted structure of the free peptide with the addition of the myristoyl (Myr) or acetyl (Ac) moiety closely resembled the protein structure that it was designed to mimic. Myr-peptide and Ac-peptide activated AMPK in muscle cells and led to reduced adipose tissue weight, body weight, blood glucose levels, insulin levels, and insulin resistance index, as expected from AMPK activation. In addition, triglyceride, cholesterol, leptin, and adiponectin levels were also lower, suggesting increased adipose tissue breakdown, a result of AMPK activation. On the other hand, liver weight and liver lipid content increased due to fat retention.We could not find an elevated pAMPK:AMPK ratio in the liver in vivo or in hepatocytes ex vivo, suggesting that the peptide does not lead to AMPK activation in hepatocytes. The finding that an AMPK-derived peptide leads to the activation of AMPK in muscle cells and in adipose tissue and leads to reduced glucose levels in obese mice, but to fat accumulation in the liver, demonstrates the differential effect of AMPK modulation in various tissues.