Acute intestinal ischemia reperfusion (IR) injury is often associated with intestinal epithelial barrier (IEB) dysfunction. Enteric glial cells (EGCs) play an essential role in maintaining the integrity of IEB functions. However, the precise mechanism of EGCs under IR stimulation remains unclear. Here, we report that EGCs are closely involved in the modulation of IEB functions in response to IR challenge. The intestinal IR treatment led to the significant upregulation of the EGC activation marker, glial fibrillary acidic protein, accompanied by the increasing abundance of glial-derived neurotrophic factor (GDNF) and inducible nitric oxidase (iNOS) proteins, which was also confirmed in in vitro hypoxia reoxygenation (HR) tests. Co-culturing with EGCs attenuated the tight junctional abnormalities, blocked the downregulation of ZO-1 and occludin protein expression, and relieved the decrease of permeability of intestinal epithelial cell (IEC) monolayers under HR treatment. Furthermore, exogenous GDNF administration displays the barrier-protective effects similar to EGCs against HR stimulation, while RNA interference-mediated knockdown of GDNF significantly inhibited the protective capability of EGCs. The expression of both GDNF and iNOS proteins of EGCs was significantly upregulated by co-culturing with IECs, which was further increased by HR treatment. Interestingly, through inhibiting iNOS activity, the barrier-protective effect of EGCs was influenced in normal condition but enhanced in HR condition. These results suggest that GDNF plays an important role in the barrier-protective mechanism of activated EGCs under IR stimulation, whereas EGCs (via iNOS release) are also involved in intestinal inflammation response, which may contribute to IEB damage induced by IR injury.