Objective: The present study was aimed to investigate the regulatory effect of Protein Kinase C (PKC) on intracellular Ca²⁺ handling in hydrogen sulfide (H₂S)- preconditioned cardiomyocytes and its consequent effects upon ischemia challenge. Methods: Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (a H₂S donor, 10^-4 M) preconditioning (SP, 30 min). Intracellular Ca²⁺ was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia/reperfusion insults to indicate the extent of hypercontracture. Result: SP motivated translocation of PKC, and to membrane fraction, but only translocation of PKC and was abolished by an ATP-sensitive potassium (K_ATP) channel blocker, glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine- induced intracellular [Ca²⁺] transients, which were reversed by a selective PKC inhibitor, chelerythrine. These data suggest that SP facilitated Ca²⁺ removal via both accelerating uptake of Ca²⁺ into sarcoplasmic reticulum (SR) and enhancing Ca²⁺ extrusion through Na⁺/ Ca²⁺ exchanger (NCX) in a PKC-dependant manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca²⁺ overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. Conclusion: We demonstrate for the first time that SP activates PKC, and in cardiomyocytes via different signaling mechanism. Such PKC activation, in turn, protects the heart against ischemia/reperfusion insults at least partly by ameliorating intracellular Ca²⁺ handling.