In this study, glial cell-derived neurotrophic factor GDNF was used as an evaluation index to explore the effect of dexmedetomidine, a commonly used clinical anesthetic and sedative, on the activation of intestinal glial cells. Rat intestinal glial cells were selected for in vitro experiments, cells were treated with dexmedetomidine at various concentrations for 24 h. The expression of the glial fibrillary acidic protein (GFAP) was determined by immunofluorescence assay. The mRNA and protein levels of GDNF were measured by RT-qPCR and Western Blot, respectively. Immunofluorescence of GFAP results indicated that intestinal glial cells had slender synapses distributed on the edge of the cell body. No differences in GAFP expression were observed between dexmedetomidine treated and untreated cells. However, dexmedetomidine treatment increased the GDNF expression significantly at both mRNA and protein levels in intestinal glial cells. Dexmedetomidine can activate intestinal glial cells and increase the secretion of glial cell-derived neurotrophic factor (GDNF).