We demonstrated here that an initial treatment with aconitine- or barium-chloride-induced arrhythmias and resulted in reduced susceptibility of the heart to the induction of arrhythmias by a repeated drug treatment 24 h after the initial one, a delayed preconditioning cardioprotection. This delayed preconditioning was accompanied by enhanced expression of cardiac muscarinic M(3) receptor and abolished by M(3)-selective antagonist. We conclude that muscarinic M(3) receptors might play an important role in conferring the pharmacological preconditioning against arrhythmias. This study thus expands our understanding of the cellular function and pathophysiological roles of muscarinic M(3) receptor and reconsolidates our view of cardioprotective effects of muscarinic M(3) receptor on myocardium.