Beneficial effects of red wine polyphenols on cardiovascular health are well known. The aim of our research was an interaction study of four red wine polyphenols – resveratrol (R), quercetin (Q), kaempferol (KF) and isorhamnetin (IR) of their ability to activate endothelial NO synthase (eNOS) promoter when used alone and in equimolar mixtures. To determine their activity, we performed a luciferase reporter gene assay on EA.hy926 cells stably transfected with a luciferase reporter gene construct containing eNOS promoter. The Bradford assay was also performed to account the cytotoxicity and/or the cell number differences. The median effect equation, as an interaction analysis evaluating synergy or antagonism of the combinations was done according to mass-action law principle. Isobolographic method was performed on selected double mixtures and dose reduction index was calculated for all mixtures. All single polyphenols activated eNOS promoter. The EC50 values were in micromolar concentrations ranging from 3.44 μM (R2 = 0.96) for kaempferol to 9.89 μM for isorhamnetin (R2 = 0.94). All mixtures activated eNOS promoter, but their interactions varied from synergy (Q+R, Q+IR+KF, Q+R+KF and Q+R+IR+KF), through additive (R+IR+KF) to antagonistic interaction (R+IR, R+KF, Q+IR, Q+KF, IR+KF and R+Q+IR). In this study, we show for the first time that red wine polyphenols activated eNOS promoter when used alone and in mixtures with different type of interactions.