Diabetes patients tend to have the gastrointestinal motility disorder. Although the relationship between the motility disorder and both the neurons and Cajal cells in the enteric nervous system (ENS) is well established, little is known about the role of enteric glial cells (EGCs) in gastric motility in diabetes. This study aimed to examine the expression of the glial marker S100B and morphology of EGCs in gastric tissues and the relationship between activated EGCs and the damage of gastric emptying in diabetic models. The diabetic model of rat was induced with 1% streptozotocin (STZ). The model rats at 7-14 days and at 56-63 days were defined as early diabetic rats and advanced diabetic rats, respectively, and normal rats at the two time periods served as their corresponding controls. The gastric emptying rate of the rats was tested by using the phenol red solution. The ultrastructure of EGCs in the gastric antrum was observed by the transmission electron microscopy, and the expression of S100B in the myenteric plexus was immunohistochemically detected. The results showed that the gastric emptying rate was significantly increased in the early diabetic rats and decreased in the advanced diabetic rats when compared with their corresponding control rats (P<0.01 for both). The ultrastructure of EGCs was mostly normal in both the early diabetic and control groups. Vacuolization of mitochondria and expansion of endoplasmic reticulum occurred in both the advanced diabetic group and its control group, and even the structure of smooth muscle cells and intestinal neurons was destroyed in the advanced diabetic group. The expression level of S100B in the advanced diabetic group was significantly decreased compared with its control group (P<0.05). It was obviously increased in the early diabetic control group when compared with the advanced diabetic control group (P<0.05). However, there was no significant difference in the S100B expression between the early diabetic group and its control group (P>0.05). The findings suggested that the gastric motility dysfunction in diabetes may be associated with the changes of morphology and number of EGCs in the myenteric plexus.