Cardio-protective effect of hydrogen sulfide (H₂S) against myocardial ischemia reperfusion injury (I/R) via preservation of mitochondria is well documented. But the distinct role of exogenous and endogenous H₂S in cardio-protection and its dependency on functional cardiac mitochondria is not understood. The present study was designed to investigate the role of exogenous H₂S preconditioning on cardiac mitochondrial subpopulation namely interfibrillar (IFM) and subsarcolemmal (SSM), in attenuating I/R injury in an isolated rat heart model in the absence of endogenous H₂S production. The well-known inhibitor of endogenous H₂S production DL-propargylglycine (cystathionine gamma lyase inhibitor) used for this purpose. Our previous studies revealed that NaSH (a H₂S donor) preconditioning at a dose of 20μM significantly reduced the infarct size and preserved the functional activities of IFM. However, this protective effect significantly declined, when the heart preconditioned with NaSH in the presence of PAG. Apparently, cardiac recovery was improved hemodynamically to a minimal level by increased concentration of NaSH (40μM). The rat heart perfused with 2, 4 dinitrophenol a mitochondrial uncoupler, before NaSH preconditioning to understand the mitochondrial dependency of exogenous NaSH, we found that cardio-protective effect of NaSH was negated even with higher concentration of H₂S. The result indicates that exogenous NaSH mediated cardio protection depends on functional cardiac mitochondria. However, no functional difference between the subpopulation i.e., interfibrillar and subsarcolemmal mitochondria observed with NaSH preconditioning in the presence of PAG.