Hydrogen sulfide (H(2)S) has been established to regulate mitochondrial respiration and ATP production, but whether the regulation is through S-sulfhydration (-SSH) of mitochondrial complexes is not well understood. Recently, H(2)S is known to exert diverse and dose-dependent effects on mitochondrial complexes. However, the involvement of S-sulfhydration of each mitochondrial complex and the activities in diabetic hearts have not been revealed. Here, we conducted comprehensive investigations into S-sulfhydration and the activities of mitochondrial complexes I-V in normal and Streptozotocin (STZ)-induced type 1 diabetic (DM) heart mitochondria. Results showed that proteins of H(2)S-producing enzymes were downregulated in DM heart mitochondria, which was accompanied by reduced mitochondrial membrane potential (MMP), greater ROS, and lower complex I and V activities, reduced complex V-SSH in DM. In both groups, supplementation with the H(2)S donor NaHS increased the S-sulfhydration of all mitochondrial complexes, and the activities of complexes I-III and V were significantly increased but complex IV activity was reduced. Consequently, mitochondrial MMP, ROS, and ATP production were normalized with NaHS in DM, whereas inhibition of H(2)S generation increased mitochondrial ROS and reduced MMP via reducing complex activities in both groups. Ischemic reperfusion did not affect NaHS-increment of S-sulfhydration of complexes I-V, but significantly impaired complex V activity in DM. Collectively, H(2)S-dependent S-sulfhydration of mitochondrial complexes I-V in normal and DM heart mitochondria were involved in the activation of mitochondrial complexes I-III/V and the inhibition of complex IV, which control cardiac mitochondrial respiration and ATP production.