Objective To observe the effects of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) on glial cell line-derived neurotrophic factor (GDNF) and its receptors in rat intestinal smooth muscle cells (ISMCs). Methods ISMCs were isolated by modified enzyme digestion in SD rats, and the positive rates of smooth muscle α-actin (α-SMA) and desmin were observed by immunocytochemical staining. ISMCs were treated with IL-1β and TNF-α for 48 hours alone or in combination. The relative mRNA expression of GDNF, receptor tyrosine kinase (Ret) and GDNF family receptor alpha-1 (GFRα1) were measured by real-time quantitative PCR. Results High-purity ISMCs were obtained by modified enzyme digestion, and over 95% of the cells expressed α-SMA and desmin. Compared with the control group, TNF-α could promote the expression of GDNF and inhibit GFRα1, but had no effects on Ret. In contrast, IL-1β could inhibit Ret but had no effects on GDNF and GFRα1. Notably, when IL-1β and TNF-α were combined, the expression of GDNF was up-regulated, while RET and GFRα1 were down-regulated. Conclusion IL-1β and TNF-α can up-regulate the expression of GDNF and down-regulate its receptors in rat ISMCs.