This study examined calcium (Ca²⁺) handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O₂ continuously for 6 hours daily from 3, 7, 14 days. In isolated-perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day-group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca²⁺ level, amplitude, time to peak and the decay time of both electrically- and caffeine-induced Ca²⁺ transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day-CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca²⁺ handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of 45Ca²⁺ flux of SR-Ca²⁺-ATPase (SERCA), ryanodine receptor (RyR) and sarcolemmal Na⁺/Ca²⁺ exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca²⁺-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5µM KT5720 or 0.5µM PKI14-22) and PKC inhibitor (5µM chelerythrine chloride or 0.2µM calphostin C) but not by Ca²⁺/calmodulin-dependent protein kinase II inhibitor KN-93 (1µM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca²⁺ handling with augmented RyR and NCX activities via protein kinase activation.