Bone marrow mesenchymal stem cells (BMSCs) have been shown to be multipotent cells that possess high self-replicating capacity. The purpose of our study was to investigate the feasibility of using enteric neuron-like cells obtained by in vitro induction and differentiated from rat BMSCs for the treatment of Hirschsprung's disease (HD). Glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) are neurotrophic factors that play important roles in neuronal development, differentiation, survival and function. Meanwhile, GDNF mutations are a major cause of HD. In this study, BMSCs were transfected with eukaryotic expression plasmids co-expressing GDNF and NT-3, and the transfected cells displayed neuron-like changes after differentiation induced by fetal gut culture medium (FGCM). Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5, VIP and nNOS. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of GDNF and NT-3 in transfected BMSCs. The present study indicates that genetically modified BMSCs co-expressing GDNF and NT-3 are able to differentiate into enteric neuronal cells and express enteric nerve markers when induced by FGCM. This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders, such as HD.