Inhibition of oxidative stress has been reported to be involved in the cardioprotective effects of hydrogen sulfide (H2S) during ischemia/reperfusion (I/R). However, the mechanism whereby H2S regulates the level of cardiac reactive oxygen species (ROS) during I/R remains unclear. Therefore, we investigated the effects of H2S on pathways that generate and scavenge ROS. Our results show that pretreating rat neonatal cardiomyocytes with NaHS, a H2S donor, reduced the levels of ROS during the hypoxia/reoxygenation (H/R) condition. We found that H2S inhibited mitochondrial complex IV activity and increased the activities of superoxide dismutases (SODS), including Mn-SOD and CuZn-SOD. Further studies indicated that H2S up-regulated the expression of Mn-SOD but not CuZn-SOD. Using a cell-free system, we showed that H2S activates CuZn-SOD. An isothermal titration calorimetry (ITC) analysis indicated that H2S directly interacts with CuZn-SOD. Taken together, H2S inhibits mitochondrial complex IV and activates SOD to decrease the levels of ROS in cardiomyocytes during I/R. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.