Objective— To determine the functional significance of physiological reactive oxygen species (ROS) levels in endothelium-dependent nitric oxide (NO)-mediated coronary vasodilatation. Methods and Results— Endothelium-derived NO is important in regulating coronary vascular tone. Excess ROS have been shown to reduce NO bioavailability, resulting in endothelial dysfunction and coronary diseases. NADPH oxidase is a major source of ROS in endothelial cells (ECs). By using lucigenin-based superoxide production and dichlorfluorescein diacetate (DCFH-DA) fluorescence-activated cell sorter assays, we found that mouse heart ECs from NADPH oxidase-knockdown (p47 phox−/− ) animals have reduced NADPH oxidase activity (>40%) and ROS levels (>30%) compared with wild-type mouse heart ECs. Surprisingly, a reduction in ROS did not improve coronary vasomotion; rather, endothelium-dependent vascular endothelial growth factor-mediated coronary vasodilatation was reduced by greater than 50% in p47 phox−/− animals. Western blots and l -citrulline assays showed a significant reduction in Akt/protein kinase B (PKB) and endothelial NO synthase phosphorylation and NO synthesis, respectively, in p47 phox−/− coronary vessels and mouse heart ECs. Adenoviral expression of constitutively active endothelial NO synthase restored vascular endothelial growth factor-mediated coronary vasodilatation in p47 phox−/− animals. Conclusion— Endothelium-dependent vascular endothelial growth factor regulation of coronary vascular tone may require NADPH oxidase-derived ROS to activate phosphatidylinositol 3-kinase-Akt-endothelial NO synthase axis.