Risk of insulin resistance, impaired glycemic control, and cardiovascular disease is excessive in overweight and obese populations. We hypothesized that increasing expression of glyoxalase 1 (Glo1)—an enzyme that catalyzes the metabolism of reactive metabolite and glycating agent methylglyoxal—may improve metabolic and vascular health. Dietary bioactive compounds were screened for Glo1 inducer activity in a functional reporter assay, hits were confirmed in cell culture, and an optimized Glo1 inducer formulation was evaluated in a randomized, placebo-controlled crossover clinical trial in 29 overweight and obese subjects. We found trans-resveratrol (tRES) and hesperetin (HESP), at concentrations achieved clinically, synergized to increase Glo1 expression. In highly overweight subjects (BMI >27.5 kg/m²), tRES-HESP coformulation increased expression and activity of Glo1 (27%, P < 0.05) and decreased plasma methylglyoxal (−37%, P < 0.05) and total body methylglyoxal-protein glycation (−14%, P < 0.01). It decreased fasting and postprandial plasma glucose (−5%, P < 0.01, and −8%, P < 0.03, respectively), increased oral glucose insulin sensitivity index (42 mL ⋅ min⁻¹ ⋅ m⁻², P < 0.02), and improved arterial dilatation Δbrachial artery flow-mediated dilatation/Δdilation response to glyceryl nitrate (95% CI 0.13–2.11). In all subjects, it decreased vascular inflammation marker soluble intercellular adhesion molecule-1 (−10%, P < 0.01). In previous clinical evaluations, tRES and HESP individually were ineffective. tRES-HESP coformulation could be a suitable treatment for improved metabolic and vascular health in overweight and obese populations.