Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca²⁺]_c. This was associated with the activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK). Membrane expression of fatty acid translocase (FAT/CD36) and ¹⁴C-palmitic acid uptake were augmented while membrane expression of glucose transporter subtype 4 (GLUT-4) as well as ³H-glucose uptake were reduced. Inhibition of AMPK and CaMKII prevented these arrhythmia-induced metabolic changes. Similar alterations of FA metabolism were observed in a transgenic mouse model (RacET) for spontaneous AF. Consistent with these findings samples of left atrial myocardium of patients with AF compared to matched samples of patients with sinus rhythm showed up-regulation of CaMKII and AMPK and increased membrane expression of FAT/CD36, resulting in lipid accumulation. These changes of FA metabolism were accompanied by decreased membrane expression of GLUT-4, increased glycogen content and increased expression of the pro-apoptotic protein bax. Irregular pacing of cardiomyocytes increases diastolic [Ca²⁺]_c and activation of CaMKII and AMPK resulting in lipid accumulation, reduced glucose uptake and increased glycogen synthesis. These metabolic changes are accompanied by an activation of pro-apoptotic signalling pathways.