Hydrogen sulfide (H2S) is an endogenous gas which has potent relaxant effect in vascular and nonvascular smooth muscles. In the present study, we have investigated how streptozotocin (STZ)-induced diabetes affected the relaxant effect of H2S in rat isolated thoracic aorta and mesenteric and pulmonary arteries. Diabetes was induced by IV injection of STZ (35 mg/kg). Insulin treatment was applied by using insulin implants. At the end of 4 and 12 weeks, the thoracic aorta and mesenteric and pulmonary arteries were isolated, and the relaxation responses to sodium hydrogen sulfide (NaHS), diazoxide, and acetylcholine were evaluated. The mRNA and protein levels of H2S-synthesizing enzymes were also examined by RT-PCR and Western Blot. The relaxation response to NaHS in the arteries isolated from both 4 and 12 week-diabetic rats was increased when compared with that obtained from the control group. Glibenclamide inhibited the relaxation response to NaHS in the arteries isolated from either diabetic or non-diabetic group of rats. Concurrent treatment of insulin to STZ-injected rats prevented the potentiation of the relaxant effect of NaHS in the arteries. However, acetylcholine and diazoxide-induced relaxation responses were not altered in diabetic group of rats. The mRNA and protein levels of H2S-synthesizing enzymes were also not altered in diabetic rats. STZ-induced experimental diabetes in rats resulted in the potentiation of the relaxation response to H2S in vascular tissues. The potentiated relaxation to H2S in diabetic arteries may play a role in vascular complications frequently seen in severe diabetes.