Emerging evidence suggests that enteric glial cells (EGCs) are important players in the intestinal tissue circuitry, where they control gut homeostasis, communicating with neurons and other neighboring cell types. However, the complex signalling pathways responsible for EGC functions are still largely unknown. To investigate the molecular mechanisms involved in cellular interactions and gut pathophysiology, EGC gene expression can be manipulated by transfection experiments. Here, we reported a protocol for gene knock-in and gene knock-down by transfection of EGCs with plasmid DNA and siRNA by lipofection.