We have previously shown that H2S, as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important source of hydrogen sulfide (H2S) in endothelial cells. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have adverse effect on cellular homeostasis. Given the importance of H2S as a vasorelaxant, angiogenesis stimulator and positive bioenergetic factor, here we tested whether a dysfunction of the 3-MST/H2S system develops in hyperglycemia/diabetes and studied its contribution to the development of diabetes-associated vascular complications. 3-mercaptopyruvate (3-MP, the substrate of 3-MST) displayed many characteristics of a H2S donor. Intraperitoneal 3-MP (1 mg/kg) raised plasma H2S levels in rats. 3-MP induced angiogenesis in vitro (10–1000 μM; bEnd3 cells) and in vivo (0.3–1 mg/kg; matrigel assay in mice). 3-MP also facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased the bioenergetic function of mitochondria. In vitro hyperglycemia or in vivo streptozotocin diabetes resulted in endothelial dysfunction mitochondrial dysfunction, impaired angiogenesis and reduced wound healing and was associated with a suppression of the vasodilatory, pro-angiogenic, wound-healing stimulating and bioenergetic effects of 3-MP. Systemic treatment with the lipoic acid restored the effect of 3-MP in diabetes, improving endothelial function, cellular bioenergetics and wound healing. We conclude that diabetes is associated with an impairment of the 3-MST/H2S pathway, and speculate that this may contribute to the pathogenesis of diabetic complications.