The impact of regular exercise in protecting cardiac deteriorating results of female sex hormone deprivation was evaluated by measuring changes in intracellular Ca2+ removal activity of sarcoplasmic reticulum (SR) in ovariectomized rats following 9-wk treadmill running exercise at moderate intensity. Despite induction of cardiac hypertrophy in exercised groups of both sham-operated and ovariectomized rats, exercise training had no effect on SR Ca2+ uptake and SR Ca(2+)-ATPase (SERCA) in hormone intact rat heart. However, exercise training normalized the suppressed maximum SR Ca2+ uptake and SERCA activity in ovariectomized rat heart. While exercise training normalized the leftward shift in pCa (-log[Ca2+])-SR Ca2+ uptake relation in ovariectomized rats, no effect was detected in exercised sham-operated rats. Similar phenomena were also observed on SERCA and on phospholamban (PLB) phosphorylation levels; exercise training in ovariectomized rats enhanced SERCA expression to reach the level as that in sham-operated rats, in which there were no differences in SERCA and phospho-PLB levels between sedentary and exercised groups. In addition, the reduction in phospho-Thr(17) PLB in myocardium of ovariectomized rats was abolished by exercise training. These results showed that regular exercise maintains the molecular activation of cardiac SR Ca2+ uptake under normal physiological conditions and is able to induce a protective impact on cardiac SR Ca2+ uptake in ovarian sex hormone-deprived status.