Ischemia-reperfusion injury is unavoidably caused by loss and subsequent restoration of blood flow during organ procurement, and prolonged ischemia-reperfusion injury IRI results in increased rates of delayed graft function and early graft loss. The endogenously produced gasotransmitter, hydrogen sulfide (H S), is a novel molecule that mitigates hypoxic tissue injury. The current study investigates the protective mitochondrial effects of H S during in vivo cold storage and subsequent renal transplantation (RTx) and in vitro cold hypoxic renal injury. Donor allografts from Brown Norway rats treated with University of Wisconsin (UW) solution + H S (150 μM NaSH) during prolonged (24-h) cold (4°C) storage exhibited significantly (p < 0.05) decreased acute necrotic/apoptotic injury and significantly (p < 0.05) improved function and recipient Lewis rat survival compared to UW solution alone. Treatment of rat kidney epithelial cells (NRK-52E) with the mitochondrial-targeted H S donor, AP39, during in vitro cold hypoxic injury improved the protective capacity of H S >1000-fold compared to similar levels of the nonspecific H S donor, GYY4137 and also improved syngraft function and survival following prolonged cold storage compared to UW solution. H S treatment mitigates cold IRI-associated renal injury via mitochondrial actions and could represent a novel therapeutic strategy to minimize the detrimental clinical outcomes of prolonged cold IRI during RTx.