Chronic intermittent hypobaric hypoxia (CIHH) has an effective cardiac protection against ischemia-reperfusion injury. However, the underlying mechanisms are not fully known. It has been shown that blockade of β-adrenergic receptor exerts anti-arrhythmic action and improves cardiac remodeling in ischemic myocardium. Thus we determined the influence of CIHH on β-adrenergic receptor activity in right ventricular papillary muscle of rats. We found that the action potential duration in right ventricular papillary muscle was significantly longer in CIHH rats than in control rats. Activation of β-adrenergic receptor with dl-isoproterenol dose-dependently increased action potential duration and the contractility in right ventricular papillary muscle. In CIHH rats, the prolonged effect of dl-isoproterenol on action potential duration and the positive inotropic effect were significantly decreased compared with that in control rats. Furthermore, radioligand-binding experiments revealed that the density and affinity of β-adrenergic receptor in right ventricular myocardium was significantly lower in CIHH rats. In addition, Western blot analysis revealed that the membrane-bound G protein Gsα expression level in cardiac myocardium was significantly lower in CIHH rats than that in control rats. Collectively, these data suggest that CIHH suppresses β-adrenergic receptor action in right ventricular papillary muscle through decreasing receptor density and affinity, as well as membrane-bound Gsα. This mechanism may be involved in the cardiac protective effect of CIHH.