Creatine (Cr) plays an important role in muscle energy homeostasis by its participation in the ATP-phosphocreatine phosphoryl exchange reaction mediated by creatine kinase (CK). Since consequences of Cr depletion are incompletely understood, we assessed morphologic, metabolic and functional consequences of systemic depletion on skeletal muscle in a mouse model with deficiency of L-arginine:glycine amidino transferase (AGAT-/-), which catalyses the first step of Cr biosynthesis. In vivo magnetic resonance spectroscopy showed a near-complete absence of Cr and phosphocreatine in resting hind limb muscle of AGAT-/- mice. Compared to wild type, the inorganic phosphate/β-ATP ratio was increased four-fold, while ATP levels were reduced by nearly half. Activities of proton-pumping respiratory chain enzymes were reduced, whereas F1F0-ATPase activity and overall mitochondrial content were increased. The Cr-deficient AGAT-/- mice had a reduced grip strength and suffered from severe muscle atrophy. Electron microscopy revealed increased amounts of intramyocellular lipid droplets and crystal formation within mitochondria of AGAT-/- muscle fibres. Ischemia resulted in exacerbated decrease of pH and increased glycolytic ATP synthesis. Oral Cr administration led to rapid uptake in skeletal muscle (faster than in brain), and reversed all muscle abnormalities, revealing that the condition of the AGAT-/- mice can be switched between Cr-deficient and normal simply by dietary manipulation. Systemic creatine depletion results in mitochondrial dysfunction and intracellular energy deficiency, as well as structural and physiological abnormalities. The consequences of AGAT deficiency are more pronounced than those of muscle-specific CK deficiency, which suggests a multifaceted involvement of creatine in muscle energy homeostasis in addition to its role in the PCr-CK system.