Skeletal muscle is one of the main physiological targets of insulin, a hormone that triggers a complex signaling cascade and that enhances the production of reactive oxygen species (ROS) in different cell types. ROS, currently considered second messengers, produce redox modifications in proteins such as ion channels that induce changes in their functional properties. In myotubes, insulin also enhances calcium release from intracellular stores. In this work, we studied in myotubes whether insulin stimulated ROS production and investigated the mechanisms underlying the insulin-dependent calcium increase: in particular, whether the late phase of the Ca2+ increase induced by insulin required ROS. We found that insulin stimulated ROS production, as detected with the probe 2',7'-dichlorofluorescein diacetate (CM-H2DCFDA). We used the translocation of p47phox from the cytoplasm to the plasma membrane as a marker of the activation of NADPH oxidase. Insulin-stimulated ROS generation was suppressed by the NADPH oxidase inhibitor apocynin and by small interfering RNA against p47phox, a regulatory NADPH oxidase subunit. Additionally, both protein kinase C and phosphatidylinositol 3-kinase are presumably involved in insulin-induced ROS generation because bisindolylmaleimide, a nonspecific protein kinase C inhibitor, and LY290042, an inhibitor of phosphatidylinositol 3-kinase, inhibited this increase. Bisindolylmaleimide, LY290042, apocynin, small interfering RNA against p47phox, and two drugs that interfere with inositol 1,4,5-trisphosphate-mediated Ca2+ release, xestospongin C and U73122, inhibited the intracellular Ca2+ increase produced by insulin. These combined results strongly suggest that insulin induces ROS generation trough NADPH activation and that this ROS increase is required for the intracellular Ca2+ rise mediated by inositol 1,4,5-trisphosphate receptors.