Resveratrol is a plant-derived phytoalexin with antioxidant, anti-inflammatory and cardio-protective properties and may be a promising therapeutic intervention strategy in cardiovascular disease. Here, we investigated the acute direct effects of trans-resveratrol (RV), on acetylcholine (ACh)-induced and flow-mediated dilation (FMD) of isolated pressurized femoral arteries of young (4-month-old) and old (26-month-old) mice. Vessel exposure to RV enhanced ACh (0.01-1.0 mM)-induced dilation (p < 0.05), but not FMD (@ 5-10 μL⋅min-1) (p < 0.05) in both young and old mice. After RV incubation, acute nitric oxide (NO) production by cultured endothelial cells was increased in response to 0.01 mM ACh, but reduced by flow (5-10 μL⋅min-1; p < 0.05). In isolated femoral arteries from endothelial nitric oxide synthase knockout (eNOS-/-) mice, RV had no overall effect on FMD, but potentiated ACh induced dilation, that was completely abolished by potassium channel blockers, Apamin and Tram 34 (p < 0.01). We demonstrate that the non-metabolised form of RV stimulates ACh-induced dilation via the NO and EDHF pathways, but not FMD by interaction with the cyclo-oxygenase pathway. Our findings have important implications in the use of RV (for both young and aged) under 'normal' non-diseased physiological states.