We previously reported that in mesenteric arteries from aged Otsuka Long-Evans Tokushima Fatty rats (OLETF) (a type 2 diabetic model), the endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired, while the endothelium-derived contracting factor (EDCF)-mediated contraction is enhanced (Am J Physiol Heart Circ Physiol 293: H1480-H1490, 2007). Here, we investigated whether acute and/or chronic treatment with metformin might improve this imbalance between the effects of the above endothelium-derived factors in mesenteric arteries isolated from OLETF rats. In acute studies on OLETF mesenteric arteries, the ACh-induced relaxation was impaired and the relaxation became weaker at high ACh concentrations. Both metformin and AICAR (an AMP-kinase (AMPK) activator, which is also activated by metformin): (a) diminished the tendency for the relaxation to reverse at high ACh concentrations, and (b) suppressed both the ACh-induced EDCF-mediated contraction and the ACh-stimulated production of prostanoids (TXA₂ and PGE₂). In studies on OLETF arteries from chronically treated animals, metformin treatment (300 mg/kg/day for 4 weeks): (a) improved the ACh-induced NO- or EDHF-mediated relaxation and COX-mediated contraction, (b) reduced the EDCF-mediated contraction, (c) suppressed the production of prostanoids, and (d) reduced superoxide generation. Metformin did not alter the protein expressions of eNOS, phospho-eNOS (Ser1177), or COX1, but it increased COX2 protein. These results suggest that metformin improves endothelial functions in OLETF mesenteric arteries by suppressing vasoconstrictor prostanoids and by reducing oxidative stress. Our data suggest that within the time-scale studied here, metformin improves endothelial function through this direct mechanism, rather than by improving metabolic abnormalities.