Type 2 diabetes mellitus (T2DM) is associated with microvascular dysfunction. We hypothesized that increased poly(ADP-ribose) polymerase 1 (PARP-1) activity contributes to microvascular dysfunction in T2DM. T2DM (db(-)/db(-)) and nondiabetic control (db(-)/db(+)) mice were treated with 2 different PARP-1 inhibitors (INO-1001, 5 mg/kg per day and ABT-888, 15 mg/kg per day) for 2 weeks. Isolated coronary arterioles were mounted in an arteriograph. Pressure-induced myogenic tone was significantly potentiated, whereas endothelium-dependent relaxation was significantly attenuated in diabetic mice compared with control mice. These results were associated with decreased endothelial NO synthase phosphorylation and cGMP level and increased PARP-1 activity in coronary arterioles from diabetic mice compared with control mice. Interestingly, PARP-1 inhibitors significantly reduced the potentiation of myogenic tone, improved endothelium-dependent relaxation, restored endothelial NO synthase phosphorylation and cGMP, and attenuated cleaved PARP-1. These results were supported by in vitro studies indicating that downregulation of PARP-1 in mesenteric resistance arteries using PARP-1 short hairpin RNA lentiviral particles significantly improved endothelium-dependent relaxation in mesenteric resistance arteries from diabetic mice compared with control mice. The inhibition of NO synthesis by N(G)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the endothelium-dependent relaxation in coronary arterioles and mesenteric resistance arteries from control and diabetic mice treated with PARP-1 inhibitors and PARP-1 short hairpin RNA lentiviral particles. In addition, we demonstrated that enhanced cleaved PARP-1, its binding to DNA, and DNA damage were reduced after PARP-1 inhibition in cultured endothelial cells stimulated with high glucose. We provide evidence that T2DM impairs microvascular function by an enhanced PARP-1 activity-dependent mechanism. Therefore, PARP-1 could be a potential target for overcoming diabetic microvascular complications.