Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.