Chronic intermittent hypobaric hypoxia (CIHH) has been shown to attenuate intracellular Na+ accumulation and Ca2+ overload during ischemia and reperfusion (I/R), both of which are closely related to the outcome of myocardial damage. Na/K pump plays an essential role in maintaining the equilibrium of intracellular Na+ and Ca2+ during I/R. It has been shown that enhancement of Na/K pump activity by ischemic preconditioning may be involved in the cardiac protection. Therefore, we tested whether Na/K pump was involved in the cardioprotection by CIHH. We found that Na/K pump current in cardiac myocytes of guinea pigs exposed to CIHH increased 1.45-fold. The K 1 and f 1, which reflect the portion of α1-isoform of Na/K pump, dramatically decreased or increased, respectively, in CIHH myocytes. Western blot analysis revealed that CIHH increased the protein expression of the α1-isoform by 76%, whereas the protein expression of the α2-isoform was not changed significantly. Na/K pump current was significantly suppressed in simulated I/R, and CIHH preserved the Na/K pump current. CIHH significantly improved the recovery of cell length and contraction during reperfusion. Furthermore, inhibition of Na/K pump by ouabain attenuated the protective effect afforded by CIHH. Collectively, these data suggest that the increase of Na/K pump activity following CIHH is due to the upregulating α1-isoform of Na/K pump, which may be one of the mechanisms of CIHH against I/R-induced injury.