Background Enteric glial cells (EGCs) play important roles in enteric integrity and regulation of gastrointestinal function. However, whether EGCs undergo pathophysiological changes in stress‐associated gastrointestinal disorders is unknown. We investigated structural and functional alterations in colonic EGCs and their roles in colonic contraction in an irritable bowel syndrome (IBS) model. Methods As a chronic stress, male Wistar rats underwent 3‐h maternal separation during postnatal days 2–14. As an acute stress, we used water‐immersion stress (4 h) in adulthood (at 8 weeks). We quantitatively and morphologically evaluated enteric neurons and EGCs using whole‐mount longitudinal muscle‐myenteric plexus preparations. Colonic contraction was analyzed with electrical field stimulation (EFS). Key Results Glial fibrillary acidic protein (GFAP) expression and the number of total, cholinergic, and nitrergic neurons were unchanged in maternally separated rats with acute stress (combined stress: an IBS model) compared with controls. However, the density of GFAP‐positive EGC processes that apparently overlapped with the neurons and the extent of bulbous swelling of terminals increased according to the stress intensity: control, acute stress, maternal separation, and combined stress. EFS‐induced colonic contractions were significantly greater in the combined stress rats than in controls. Higher dose of fluorocitrate, a selective inhibitor of EGC metabolism, was required to inhibit both EFS‐induced contraction and EGCs activation in the combined stress rats than in controls. Conclusions & Inferences Colonic EGCs exhibited structural alterations according to the stress intensity. EGCs were associated with stress‐induced colonic hyper‐contraction in the combined stress rats, which may underlie the pathogenesis of IBS. Enteric glial cells (EGCs) play important roles in the regulation of gastrointestinal function, but manifestations of colonic EGCs in irritable bowel syndrome (IBS) patients remain unknown. EGCs exhibited hyperplasia of their processes that apparently overlapped with the neurons and changes in bulbous swelling of terminals, and were also associated with stress‐induced hyper colonic contraction in the maternally separated rats. Such changes in EGCs may underlie the pathogenesis of IBS.