Obese subjects with the metabolic syndrome (MS) are more prone to microvascular complications than obese subjects without the metabolic syndrome (MS). Excessive vascular nitric oxide (NO) production has been demonstrated in MS compared to MS, perhaps driven by increased inflammation or oxidative stress. We tested whether in MS, folic acid (FA) treatment could normalize NO synthase (NOS)-dependence of vascular tone in the retina and kidney. MS (n = 49) and MS (n = 26) subjects were included in a randomized, double-blind, crossover trial. After 4-weeks' treatment with placebo or FA (5mg/day), several cytokines (C-reactive protein (CRP), interleukin-1Β, adiponectin), and markers of oxidative stress (glutathione/oxidized glutathione (GSH/GSSG) ratio, total antioxidant capacity (TAC)) were determined. NOS-dependence of retinal and renal vascular tone was assessed by retinal scanning laser Doppler flowmetry and renal clearance technique, respectively. FA had no effect on cytokine levels, but increased GSH/GSSG ratio overall (36 76 vs. 102 200, P = 0.04), indicative of a reduction in oxidative stress. In MS, treatment with FA reduced NOS-dependence of retinal and renal vascular tone compared to placebo (P = 0.03 and P = 0.04, respectively). FA had no effect in MS. After treatment with FA, NOS-dependence of retinal and renal vascular tone was similar between MS and MS. Retinal and renal vascular tone in MS subjects is characterized by increased dependence on NOS. NOS-dependence in MS could be corrected by FA treatment to levels not dissimilar in MS, and this was associated with a reduction in oxidative stress. Future trials should test whether these effects translate into a reduction of microvascular complications.