Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor cloned from mouse brain tissue by Lin et al. (1993) and can also be secreted by Sertoli cells. GDNF has a variety of physiological functions and plays important roles in the nervous system and in the reproduction of spermatogonial stem cells (SSCs). To date, commercialized murine and human GDNF have typically been used for the in vitro culture of SSCs from domestic animals. However, whether endogenous GDNF is superior to xenogenic GDNF in SSC culture in vitro remains unclear. To answer this question, the buffalo GDNF gene was cloned and integrated into the PiggyBac eukaryotic expression vector, which was stably expressed in Sertoli cells, thus establishing a PB-GDNF-Sertoli cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the GDNF gene expression in the PB-GDNF-Sertoli cell line was significantly increased compared with that in the control Sertoli cell line (p<0.01). Enzyme-linked immunosorbent assay (ELISA) results showed that GDNF gene secretion was also significantly increased in PB-GDNF-Sertoli cells compared with that in control Sertoli cells (p<0.05). We further compared two types of spermatogonia cultured on either PB-GDNF-Sertoli cells or control Sertoli cells, revealing that the use of PB-GDNF-Sertoli cells as a feeder layer could significantly increase the expression levels of DDX4 (p<0.01), PLZF (p<0.01) and NANOS2 (p<0.05) in buffalo spermatogonia in vitro. These results suggested that overexpression of GDNF could effectively maintain the pluripotency of buffalo spermatogonia in vitro, laying the foundation for improving the in vitro culture of buffalo spermatogonia.