Mitochondrial biogenesis is a critical adaptation to chronic energy deprivation, yet the signaling mechanisms responsible for this response are poorly understood. To examine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved fuel sensor, in mitochondrial biogenesis we studied transgenic mice expressing a dominant-negative mutant of AMPK in muscle (DN-AMPK). Both DN-AMPK and WT mice were treated with β-guanidinopropionic acid (GPA), a creatine analog, which led to similar reductions in the intramuscular ATP/AMP ratio and phosphocreatine concentrations. In WT mice, GPA treatment resulted in activation of muscle AMPK and mitochondrial biogenesis. However, the same GPA treatment in DN-AMPK mice had no effect on AMPK activity or mitochondrial content. Furthermore, AMPK inactivation abrogated GPA-induced increases in the expression of peroxisome proliferator-activated receptor γ coactivator 1α and calcium/calmodulin-dependent protein kinase IV (both master regulators of mitochondrial biogenesis). These data demonstrate that by sensing the energy status of the muscle cell, AMPK is a critical regulator involved in initiating mitochondrial biogenesis.