Vascular endothelial growth factor (VEGF) is the most potent stimulatory factor of angiogenesis. Its expression is induced by reactive oxygen species (ROS) in hypoxic conditions and by insulin in normoxic cells. Both ROS and insulin can activate mitogen-activated protein kinases (MAPKs) and induce the transcriptional factor Sp1, components that are essential for VEGF gene expression. The aim of this study was to investigate the role of ROS producing NADPH oxidase enzymes (NOX-es) in insulin-regulated VEGF gene activation. To achieve this goal we chose HepG2 cells as our model system as these cells express the NADPH oxidase isoform NOX3 and respond to insulin stimulation with enhanced ROS production and mRNA transcription and production of VEGF. We demonstrate that in control cells insulin stimulation leads to H2O2 generation, a biphasic activation of p42/44 MAPK and the induction of both Sp1 and HIF-1alpha. Transfection of NOX3-specific siRNA abrogates H2O2 production and inhibits exclusively the second phase of p42/44 MAPK phosphorylation and Sp1 DNA binding and thus prevents upregulation of VEGF-A mRNA expression. In conclusion, our results demonstrate that NOX3, a ROS generating NADPH oxidase, plays an integral role in insulin-induced p42/44 MAPK signal transmission and VEGF-A production.