Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestinal epithelial cells from anoxia/reoxygenation in vitro, and to protect the intestines from ischemia/reperfusion (I/R) injury in vivo. The goal of the present study was to determine whether the cytoprotective effects of HB-EGF were due, in part, to its ability to decrease reactive oxygen species (ROS) production. Human whole blood, polymorphonuclear leukocytes, and monocytes, as well as rat intestinal epithelial cells, were exposed to stimuli designed to produce an oxidative burst in these cells. Treatment of the cells with HB-EGF led to a significant decrease in oxidative burst production. In vivo, total midgut I/R injury in rats led to increased ROS production, which was markedly decreased by HB-EGF treatment. Histochemically, I/R injury led to increased ROS production, which was significantly decreased with HB-EGF treatment. HB-EGF cytoprotection is due, in part, to its ability to decrease ROS production. Future studies will determine the mechanisms by which HB-EGF exerts these effects.