Circadian rhythms influence the incidence of adverse cardiac events but the underlying mechanisms are not well defined. We sought to investigate the role of the β3-adrenoceptor (β3-AR) in cardiac circadian disorders and arrhythmia severity after myocardial infarction (MI). MI was created by ligating the left anterior descending coronary artery of the rat heart in situ. Circadian variations of the myocardial expressions of β3-AR and clock genes Bmal1 and Npas2 were examined by real time reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Electrocardiograms and myocardial contraction were recorded in vivo and/or ex vivo. Ventricular tachyarrhythmias were induced by isoprenaline. Normal rats showed circadian oscillations in both the myocardial transcriptional expression of β3-AR and the β3-AR-induced positive chronotropic and negative inotropic cardiac effects. However, these circadian rhythms were significantly blunted or even abolished in rats with either acute MI (within 24 h) or healed MI (14 days after coronary ligation). The nocturnal level of β3-AR protein was higher in MI rats than in normal rats. In contrast, the circadian oscillations of the transcripts of Bmal1 and Npas2 in the myocardium were significantly augmented in rats with either acute MI or healed MI. BRL37344, a preferential β3-AR selective agonist, reduced the occurrence of ventricular tachycardia (VT) and ventricular fibrillation (VF) in rats with either acute MI or healed MI. We conclude that circadian rhythms of myocardial β3-AR activities are disturbed after MI and β3-AR activation offers anti-arrhythmic protection.