Enteric glia play fundamental roles in the plasticity and homeostasis of the enteric nervous system. Disruptions in their function contribute to intestinal dysfunction and are associated with gut pathophysiology. To further understand the role of enteric glia in gut physiology, it is essential to use a pure and reliable primary culture method. Here, we describe a protocol for the isolation, purification, enrichment, and culture of enteric glia from mature rodent intestines.