Endurance training is accompanied by important adaptations in both cardiovascular and autonomic nervous systems. Previous works have shown that the main component of gap junctions in the ventricular myocardium (connexin 43 (Cx43) can be regulated by adrenergic stimulus. On the other hand, training raises vagal and decreases sympathetic tone, while augmenting myocardial sensitivity to sympathetic stimulation during exercise. We therefore evaluated the regulation of Cx43 expression by sympathetic tone during exercise in trained and sedentary mice. Training induced an increase in the protein level of Cx43 by 45–70% under resting conditions. The expression of Cx43 was inhibited in trained but not in untrained mice in response to a 60 min exercise bout. Normal basal expression was restored after 60 min of resting. Cx43 reached a minimum that was not different from basal expression in untrained mice. In accordance, electrocardiography and action potential analysis did not reveal major electrophysiological implications for the drop in Cx43 abundance in trained-exercise mice. We prevented Cx43 inhibition using propranolol, and observed increased basal mRNA levels of β-adrenergic receptors without significant changes in the ratio β1 to β2. In conclusion, we showed that Cx43 expression is transiently inhibited by β-adrenergic stimulus in trained mice during acute exercise.