Atrial fibrillation is the most common arrhythmia encountered in clinical practice. It can cause severe complications such as congestive heart failure and stroke. However, identification of prime targets for efficient therapeutic intervention remains a challenge. In vitro rabbit heart models of ischemia-, stretch-, and cholinergic agitation-induced atrial fibrillation were developed, and pharmacological interventions of mitochondrial translocator protein (TSPO) were adopted to explore the role of the mitochondrial protein in the aforementioned atrial fibrillations. Fura-2 AM and Mg(2+)-Fura-2 AM were used to monitor the alterations of intracellular Ca(2+) and ATP respectively under chemical ischemia or cholinergic agitation. The results showed that inhibition of TSPO significantly reduced the incidence of all three types of atrial fibrillation. In addition, TSPO inhibition ameliorated the cytoplasmic Ca(2+) overload and energy compromise facing to chemical ischemia or cholinergic agitation in HL-1 cells, an atrial muscle cell line. Thus, TSPO may be an important molecule in the context of different kinds of atrial fibrillation, and a novel and common target for atrial fibrillation treatment.